Humanized antibodies that recognize beta amyloid peptide

ABSTRACT

The invention provides improved agents and methods for treatment of diseases associated with amyloid deposits of Aβ in the brain of a patient. Preferred agents include antibodies, e.g., humanized antibodies.

RELATED APPLICATIONS

This application claims the benefit of prior-filed provisional patentapplication U.S. Ser. No. 60/474,654 (filed May 30, 2003) entitled“Humanized Antibodies That Recognize Beta Amyloid Peptide”. The entirecontent of the above-referenced application is incorporated herein byreference.

BACKGROUND OF THE INVENTION

Alzheimer's disease (AD) is a progressive disease resulting in seniledementia. See generally Selkoe, TINS 16:403 (1993); Hardy et al., WO92/13069; Selkoe, J. Neuropathol. Exp. Neurol. 53:438 (1994); Duff etal., Nature 373:476 (1995); Games et al., Nature 373:523 (1995). Broadlyspeaking, the disease falls into two categories: late onset, whichoccurs in old age (65+years) and early onset, which develops well beforethe senile period, i.e., between 35 and 60 years. In both types ofdisease, the pathology is the same but the abnormalities tend to be moresevere and widespread in cases beginning at an earlier age. The diseaseis characterized by at least two types of lesions in the brain,neurofibrillary tangles and senile plaques. Neurofibrillary tangles areintracellular deposits of microtubule associated tau protein consistingof two filaments twisted about each other in pairs. Senile plaques(i.e., amyloid plaques) are areas of disorganized neuropil up to 150 μmacross with extracellular amyloid deposits at the center which arevisible by microscopic analysis of sections of brain tissue. Theaccumulation of amyloid plaques within the brain is also associated withDown's syndrome and other cognitive disorders.

The principal constituent of the plaques is a peptide termed Aβ orβ-amyloid peptide. Aβ peptide is a 4-kDa internal fragment of 39-43amino acids of a larger transmembrane glycoprotein named protein termedamyloid precursor protein (APP). As a result of proteolytic processingof APP by different secretase enzymes, Aβ is primarily found in both ashort form, 40 amino acids in length, and a long form, ranging from42-43 amino acids in length. Part of the hydrophobic transmembranedomain of APP is found at the carboxy end of Aβ, and may account for theability of Aβ to aggregate into plaques, particularly in the case of thelong form. Accumulation of amyloid plaques in the brain eventually leadsto neuronal cell death. The physical symptoms associated with this typeof neural deterioration characterize Alzheimer's disease.

Several mutations within the APP protein have been correlated with thepresence of Alzheimer's disease. See, e.g., Goate et al., Nature349:704) (1991) (valine⁷¹⁷ to isoleucine); Chartier Harlan et al. Nature353:844 (1991)) (valine⁷¹⁷ to glycine); Murrell et al., Science 254:97(1991) (valine⁷¹⁷ to phenylalanine); Mullan et al., Nature Genet. 1:345(1992) (a double mutation changing lysine⁵⁹⁵-methionine⁵⁹⁶ toasparagine⁵⁹⁵-leucine⁵⁹⁶). Such mutations are thought to causeAlzheimer's disease by increased or altered processing of APP to Aβ,particularly processing of APP to increased amounts of the long form ofAβ (i.e., Aβ 1-42 and Aβ1-43). Mutations in other genes, such as thepresenilin genes, PS1 and PS2, are thought indirectly to affectprocessing of APP to generate increased amounts of long form Aβ (seeHardy, TINS 20: 154 (1997)).

Mouse models have been used successfully to determine the significanceof amyloid plaques in Alzheimer's (Games et al., supra, Johnson-Wood etal., Proc. Natl. Acad. Sci. USA 94:1550 (1997)). In particular, whenPDAPP transgenic mice, (which express a mutant form of human APP anddevelop Alzheimer's disease at a young age), are injected with the longform of Aβ, they display both a decrease in the progression ofAlzheimer's and an increase in antibody titers to the Aβ peptide (Schenket al., Nature 400, 173 (1999)). The observations discussed aboveindicate that Aβ, particularly in its long form, is a causative elementin Alzheimer's disease.

Accordingly, there exists the need for new therapies and reagents forthe treatment of Alzheimer's disease, in particular, therapies andreagents capable of effecting a therapeutic benefit at physiologic(e.g., non-toxic) doses.

SUMMARY OF THE INVENTION

The present invention features new immunological reagents, inparticular, therapeutic antibody reagents for the prevention andtreatment of amyloidogenic disease (e.g., Alzheimer's disease). Theinvention is based, at least in part, on the identification andcharacterization of a monoclonal antibody, 12A11, that specificallybinds to Aβ peptide and is effective at reducing plaque burdenassociated with amyloidogenic disorders. Structural and functionalanalysis of this antibody leads to the design of various humanizedantibodies for prophylactic and/or therapeutic use. In particular, theinvention features humanization of the variable regions of this antibodyand, accordingly, provides for humanized immunoglobulin or antibodychains, intact humanized immunoglobulins or antibodies, and functionalimmunoglobulin or antibody fragments, in particular, antigen bindingfragments, of the featured antibody.

Polypeptides comprising the complementarity determining regions (CDRs)of the featured monoclonal antibody are also disclosed, as arepolynucleotide reagents, vectors and host cells suitable for encodingsaid polypeptides.

Methods for treating amyloidogenic diseases or disorders (e.g.,Alzheimer's disease) are disclosed, as are pharmaceutical compositionsand kits for use in such applications.

Also featured are methods of identifying residues within the featuredmonoclonal antibodies which are important for proper immunologicfunction and for identifying residues which are amenable to substitutionin the design of humanized antibodies having improved binding affinitiesand/or reduced immunogenicity, when used as therapeutic reagents.

Also featured are antibodies (e.g., humanized antibodies) having alteredeffector functions, and therapeutic uses thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 graphically depicts results from an experiment which examines theeffectiveness of various antibodies, including 12A11, at clearing Aβplaques in an ex vivo phagocytosis assay.

FIG. 2A graphically depicts results from an experiment which examinesthe effectiveness of various antibodies, including 12A11, at reducingtotal Aβ levels. The bars represent median values, and the dashedhorizontal line indicates the control level. FIG. 2B graphically depictsresults from an experiment which analyzes the effectiveness of variousantibodies, including 12A11, at reducing neuritic dystrophy. The barsrepresent median values, and the dashed horizontal line indicates thecontrol level. Data are shown for individual animals and expressed asthe percentage of neuritic dystrophy relative to the mean of the control(set at 100%).

FIG. 3A depicts a DNA sequence including the murine 12A11 VL chainsequence and the deduced amino acid sequence for the VL chain (SEQ IDNOs: 5 and 2, respectively). Mature VL chain is indicated by a solidblack bar. CDRs are indicated by open bars. FIG. 3B depicts a DNAsequence including the murine 12A11 VH chain sequence and the deducedamino acid sequence for the VH chain (SEQ ID NOs: 6 and 3,respectively). Mature VH chain is indicated by a solid black bar. CDRsare indicated by open bars. DNA sequences include cloning sites andKozak seqeunces (upstream of coding sequences) and splice and cloningsequences (downstream).

FIG. 4 graphically depicts the ELISA results from an experimentmeasuring the binding of chimeric 12A11, chimeric and humanized 3D6, andchimeric and humanized 12B4 to Aβ 1-42.

FIG. 5A depicts an alignment of the amino acid sequences of the lightchain of murine (or chimeric) 12A11 (SEQ ID NO:2), humanized 12A11(mature peptide, SEQ ID NO:7), GenBank BAC01733 (SEQ ID NO: 8) andgermline A19 (X63397, SEQ ID NO: 9) antibodies. CDR regions are boxed.Packing residues are underlined. Numbered from the first methionine, notKabat numbering. FIG. 5B depicts an alignment of the amino acidsequences of the heavy chain of murine (or chimeric) 12A11 (SEQ IDNO:4), humanized 12aA11 (version 1) (mature peptide, SEQ ID NO:10),GenBank AAA69734 (SEQ ID NO:11), and germline GenBank 567123 antibodies(SEQ ID NO:12). Packing residues are underlined, canonical residues arein solid fill and vernier residues are in dotted fill. Numbered from thefirst methionine, not Kabat numbering.

FIG. 6A-B depicts an alignment of the amino acid sequences of the heavychains of humanized 12A11 v1 (SEQ ID NO:10), v2 (SEQ ID NO:13), v2.1(SEQ ID NO:14), v3 (SEQ ID NO:15), v4.1 (SEQ ID NO:16), v4.2 (SEQ IDNO:17), v4.3 (SEQ ID NO:18), v4.4 (SEQ ID NO:19), v5.1 (SEQ ID NO:20),v5.2 (SEQ ID NO:21), v5.3 (SEQ ID NO:22), v5.4 (SEQ ID NO:23), v5.5 (SEQID NO:24), v5.6 (SEQ ID NO:25), v6.1 (SEQ ID NO:26), v6.2 (SEQ IDNO:27), v6.3 (SEQ ID NO:28), v6.4 (SEQ ID NO:29), v7 (SEQ ID NO:30) andv8 (SEQ ID NO:31). FIG. 6C sets forth the backmutations made inhumanized 12A11 v1 to v8.

FIG. 7 depicts the results from an aggreated Aβ (1-42) ELISA comparingchimeric 12A11, humanized 12A11 v1, humanized 12A11 v2, humanized 12A11v2.1, and humanized 12A11 v3.

FIG. 8 depicts the results of a competitive Aβ 1-42 ELISA binding assaycomparing murine 12A11, chimeric 12A11 and 12A11 v1.

DETAILED DESCRIPTION OF THE INVENTION

The present invention features new immunological reagents and methodsfor preventing or treating Alzheimer's disease or other amyloidogenicdiseases. The invention is based, at least in part, on thecharacterization of a monoclonal immunoglobulin, 12A11, effective atbinding beta amyloid protein (Aβ) (e.g., binding soluble and/oraggregated Aβ), mediating phagocytosis (e.g., of aggregated Aβ),reducing plaque burden and/or reducing neuritic dystrophy (e.g., in apatient). The invention is further based on the determination andstructural characterization of the primary and secondary structure ofthe variable light and heavy chains of the 12A11 immunoglobulin and theidentification of residues important for activity and immunogenicity.

Immunoglobulins are featured which include a variable light and/orvariable heavy chain of the 12A11 monoclonal immunoglobulin describedherein. Preferred immunoglobulins, e.g., therapeutic immunoglobulins,are featured which include a humanized variable light and/or humanizedvariable heavy chain. Preferred variable light and/or variable heavychains include a complementarity determining region (CDR) from the 12A11immunoglobulin (e.g., donor immunoglobulin) and variable frameworkregions from or substantially from a human acceptor immunoglobulin. Thephrase “substantially from a human acceptor immunoglobulin” means thatthe majority or key framework residues are from the human acceptorsequence, allowing however, for substitution of residues at certainpositions with residues selected to improve activity of the humanizedimmunoglobulin (e.g., alter activity such that it more closely mimicsthe activity of the donor immunoglobulin) or selected to decrease theimmunogenicity of the humanized immunoglobulin.

In one embodiment, the invention features a humanized immunoglobulinlight or heavy chain that includes 12A11 variable region complementaritydetermining regions (CDRs) (i.e., includes one, two or three CDRs fromthe light chain variable region sequence set forth as SEQ ID NO:2 orincludes one, two or three CDRs from the heavy chain variable regionsequence set forth as SEQ ID NO:4), and includes a variable frameworkregion from a human acceptor immunoglobulin light or heavy chainsequence, optionally having at least one residue of the frameworkresidue backmutated to a corresponding murine residue, wherein saidbackmutation does not substantially affect the ability of the chain todirect Aβ binding.

In one embodiment, the invention features a humanized immunoglobulinlight or heavy chain that includes 12A11 variable region complementaritydetermining regions (CDRs) (i.e., includes one, two or three CDRs fromthe light chain variable region sequence set forth as SEQ ID NO:2 orincludes one, two or three CDRs from the heavy chain variable regionsequence set forth as SEQ ID NO:4), and includes a variable frameworkregion substantially from a human acceptor immunoglobulin light or heavychain sequence, optionally having at least one residue of the frameworkresidue backmutated to a corresponding murine residue, wherein saidbackmutation does not substantially affect the ability of the chain todirect Aβ binding.

In another embodiment, the invention features a humanized immunoglobulinlight or heavy chain that includes 12A11 variable region complementaritydetermining regions (CDRs) (e.g., includes one, two or three CDRs fromthe light chain variable region sequence set forth as SEQ ID NO:2 and/orincludes one, two or three CDRs from the heavy chain variable regionsequence set forth as SEQ ID NO:4), and includes a variable frameworkregion substantially from a human acceptor immunoglobulin light or heavychain sequence, optionally having at least one framework residuesubstituted with the corresponding amino acid residue from the mouse12A11 light or heavy chain variable region sequence, where the frameworkresidue is selected from the group consisting of (a) a residue thatnon-covalently binds antigen directly; (b) a residue adjacent to a CDR;(c) a CDR-interacting residue (e.g., identified by modeling the light orheavy chain on the solved structure of a homologous known immunoglobulinchain); and (d) a residue participating in the VL-VH interface.

In another embodiment, the invention features a humanized immunoglobulinlight or heavy chain that includes 12A11 variable region CDRs andvariable framework regions from a human acceptor immunoglobulin light orheavy chain sequence, optionally having at least one framework residuesubstituted with the corresponding amino acid residue from the mouse12A11 light or heavy chain variable region sequence, where the frameworkresidue is a residue capable of affecting light chain variable regionconformation or function as identified by analysis of athree-dimensional model of the variable region, for example a residuecapable of interacting with antigen, a residue proximal to the antigenbinding site, a residue capable of interacting with a CDR, a residueadjacent to a CDR, a residue within 6 Å of a CDR residue, a canonicalresidue, a vernier zone residue, an interchain packing residue, anunusual residue, or a glycoslyation site residue on the surface of thestructural model.

In another embodiment, the invention features, in addition to thesubstitutions described above, a substitution of at least one rare humanframework residue. For example, a rare residue can be substituted withan amino acid residue which is common for human variable chain sequencesat that position. Alternatively, a rare residue can be substituted witha corresponding amino acid residue from a homologous germline variablechain sequence.

In another embodiment, the invention features a humanized immunoglobulinthat includes a light chain and a heavy chain, as described above, or anantigen-binding fragment of said immunoglobulin. In an exemplaryembodiment, the humanized immunoglobulin binds (e.g., specificallybinds) to beta amyloid peptide (Aβ) with a binding affinity of at least10⁷ M⁻¹, 10⁸ M⁻¹, or 10⁹ M⁻¹. In another embodiment, the immunoglobulinor antigen binding fragment includes a heavy chain having isotype γ1. Inanother embodiment, the immunoglobulin or antigen binding fragment binds(e.g., specifically binds) to either or both soluble beta amyloidpeptide (Aβ) and aggregated Aβ. In another embodiment, theimmunoglobulin or antigen binding fragment captures soluble Aβ (e.g.,soluble Aβ1-42). In another embodiment, the immunoglobulin or antigenbinding fragment mediates phagocytosis (e.g., induces phagocytosis) ofbeta amyloid peptide (Aβ). In yet another embodiment, the immunoglobulinor antigen binding fragment-crosses the blood-brain barrier in asubject. In yet another embodiment, the immunoglobulin or antigenbinding fragment reduces either or both beta amyloid peptide (Aβ) burdenand neuritic dystrophy in a subject.

In another embodiment, the invention features chimeric immunoglobulinsthat include 12A11 variable regions (e.g., the variable region sequencesset forth as SEQ ID NO:2 or SEQ ID NO:4). In yet another embodiment, theimmunoglobulin, or antigen-binding fragment thereof, further includesconstant regions from IgG1.

The immunoglobulins described herein are particularly suited for use intherapeutic methods aimed at preventing or treating amyloidogenicdiseases. In one embodiment, the invention features a method ofpreventing or treating an amyloidogenic disease (e.g., Alzheimer'sdisease) that involves administering to the patient an effective dosageof a humanized immunoglobulin as described herein. In anotherembodiment, the invention features pharmaceutical compositions thatinclude a humanized immunoglobulin as described herein and apharmaceutical carrier. Also featured are isolated nucleic acidmolecules, vectors and host cells for producing the immunoglobulins orimmunoglobulin fragments or chains described herein, as well as methodsfor producing said immunoglobulins, immunoglobulin fragments orimmunoglobulin chains.

The present invention further features a method for identifying 12A11residues amenable to substitution when producing a humanized 12A11immunoglobulin, respectively. For example, a method for identifyingvariable framework region residues amenable to substitution involvesmodeling the three-dimensional structure of a 12A11 variable region on asolved homologous immunoglobulin structure and analyzing said model forresidues capable of affecting 12A11 immunoglobulin variable regionconformation or function, such that residues amenable to substitutionare identified. The invention further features use of the variableregion sequence set forth as SEQ ID NO:2 or SEQ ID NO:4, or any portionthereof, in producing a three-dimensional image of a 12A11immunoglobulin, 12A11 immunoglobulin chain, or domain thereof.

The present invention further features immunoglobulins having alteredeffector function, such as the ability to bind effector molecules, forexample, complement or a receptor on an effector cell. In particular,the immunoglobulin of the invention has an altered constant region,e.g., Fc region, wherein at least one amino acid residue in the Fcregion has been replaced with a different residue or side chain. In oneembodiment, the modified immunoglobulin is of the IgG class, comprisesat least one amino acid residue replacement in the Fc region such thatthe immunoglobulin has an altered effector function, e.g., as comparedwith an unmodified immunoglobulin. In particular embodiments, theimmunoglobulin of the invention has an altered effector function suchthat it is less immunogenic (e.g., does not provoke undesired effectorcell activity, lysis, or complement binding), has improved amyloidclearance properties, and/or has a desirable half-life.

Prior to describing the invention, it may be helpful to an understandingthereof to set forth definitions of certain terms to be usedhereinafter.

The term “immunoglobulin” or “antibody” (used interchangeably herein)refers to a protein having a basic four-polypeptide chain structureconsisting of two heavy and two light chains, said chains beingstabilized, for example, by interchain disulfide bonds, which has theability to specifically bind antigen. The term “single-chainimmunoglobulin” or “single-chain antibody” (used interchangeably herein)refers to a protein having a two-polypeptide chain structure consistingof a heavy and a light chain, said chains being stabilized, for example,by interchain peptide linkers, which has the ability to specificallybind antigen. The term “domain” refers to a globular region of a heavyor light chain polypeptide comprising peptide loops (e.g., comprising 3to 4 peptide loops) stabilized, for example, by β-pleated sheet and/orintrachain disulfide bond. Domains are further referred to herein as“constant” or “variable”, based on the relative lack of sequencevariation within the domains of various class members in the case of a“constant” domain, or the significant variation within the domains ofvarious class members in the case of a “variable” domain. Antibody orpolypeptide “domains” are often referred to interchangeably in the artas antibody or polypeptide “regions”. The “constant” domains of anantibody light chain are referred to interchangeably as “light chainconstant regions”, “light chain constant domains”, “CL” regions or “CL”domains. The “constant” domains of an antibody heavy chain are referredto interchangeably as “heavy chain constant regions”, “heavy chainconstant domains”, “CH” regions or “CH” domains). The “variable” domainsof an antibody light chain are referred to interchangeably as “lightchain variable regions”, “light chain variable domains”, “VL” regions or“VL” domains). The “variable” domains of an antibody heavy chain arereferred to interchangeably as “heavy chain constant regions”, “heavychain constant domains”, “VH” regions or “VH” domains).

The term “region” can also refer to a part or portion of an antibodychain or antibody chain domain (e.g., a part or portion of a heavy orlight chain or a part or portion of a constant or variable domain, asdefined herein), as well as more discrete parts or portions of saidchains or domains. For example, light and heavy chains or light andheavy chain variable domains include “complementarity determiningregions” or “CDRs” interspersed among “framework regions” or “FRs”, asdefined herein.

Immunoglobulins or antibodies can exist in monomeric or polymeric form,for example, IgM antibodies which exist in pentameric form and/or IgAantibodies which exist in monomeric, dimeric or multimeric form. Theterm “fragment” refers to a part or portion of an antibody or antibodychain comprising fewer amino acid residues than an intact or completeantibody or antibody chain. Fragments can be obtained via chemical orenzymatic treatment of an intact or complete antibody or antibody chain.Fragments can also be obtained by recombinant means. Exemplary fragmentsinclude Fab, Fab′, F(ab′)₂, Fabc and/or Fv fragments. The term“antigen-binding fragment” refers to a polypeptide fragment of animmunoglobulin or antibody that binds antigen or competes with intactantibody (i.e., with the intact antibody from which they were derived)for antigen binding (i.e., specific binding).

The term “conformation” refers to the tertiary structure of a protein orpolypeptide (e.g., an antibody, antibody chain, domain or regionthereof). For example, the phrase “light (or heavy) chain conformation”refers to the tertiary structure of a light (or heavy) chain variableregion, and the phrase “antibody conformation” or “antibody fragmentconformation” refers to the tertiary structure of an antibody orfragment thereof.

“Specific binding” of an antibody means that the antibody exhibitsappreciable affinity for a particular antigen or epitope and, generally,does not exhibit significant crossreactivity. In exemplary embodiments,the antibody exhibits no crossreactivity (e.g., does not crossreact withnon-Aβ peptides or with remote epitopes on Aβ). “Appreciable” orpreferred binding includes binding with an affinity of at least 10⁶,10⁷, 10⁸, 10⁹ M⁻¹, or 10¹⁰ M⁻¹. Affinities greater than 10⁷ M⁻¹,preferably greater than 10⁸ M⁻¹ are more preferred. Values intermediateof those set forth herein are also intended to be within the scope ofthe present invention and a preferred binding affinity can be indicatedas a range of affinities, for example, 10⁶ to 10¹⁰ M⁻¹, preferably 10⁷to 10¹⁰ M⁻¹, more preferably 10⁸ to 10¹⁰ M⁻¹. An antibody that “does notexhibit significant crossreactivity” is one that will not appreciablybind to an undesirable entity (e.g., an undesirable proteinaceousentity). For example, an antibody that specifically binds to Aβ willappreciably bind Aβ but will not significantly react with non-Aβproteins or peptides (e.g., non-Aβ proteins or peptides included inplaques). An antibody specific for a particular epitope will, forexample, not significantly crossreact with remote epitopes on the sameprotein or peptide. Specific binding can be determined according to anyart-recognized means for determining such binding. Preferably, specificbinding is determined according to Scatchard analysis and/or competitivebinding assays.

Binding fragments are produced by recombinant DNA techniques, or byenzymatic or chemical cleavage of intact immunoglobulins. Bindingfragments include Fab, Fab′, F(ab′)₂, Fabc, Fv, single chains, andsingle-chain antibodies. Other than “bispecific” or “bifunctional”immunoglobulins or antibodies, an immunoglobulin or antibody isunderstood to have each of its binding sites identical. A “bispecific”or “bifunctional antibody” is an artificial hybrid antibody having twodifferent heavy/light chain pairs and two different binding sites.Bispecific antibodies can be produced by a variety of methods includingfusion of hybridomas or linking of Fab′ fragments. See, e.g.,Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelnyet al., J. Immunol. 148, 1547-1553 (1992).

The term “humanized immunoglobulin” or “humanized antibody” refers to animmunoglobulin or antibody that includes at least one humanizedimmunoglobulin or antibody chain (i.e., at least one humanized light orheavy chain). The term “humanized immunoglobulin chain” or “humanizedantibody chain” (i.e., a “humanized immunoglobulin light chain” or“humanized immunoglobulin heavy chain”) refers to an immunoglobulin orantibody chain (i.e., a light or heavy chain, respectively) having avariable region that includes a variable framework region substantiallyfrom a human immunoglobulin or antibody and complementarity determiningregions (CDRs) (e.g., at least one CDR, preferably two CDRs, morepreferably three CDRs) substantially from a non-human immunoglobulin orantibody, and further includes constant regions (e.g., at least oneconstant region or portion thereof, in the case of a light chain, andpreferably three constant regions in the case of a heavy chain). Theterm “humanized variable region” (e.g., “humanized light chain variableregion” or “humanized heavy chain variable region”) refers to a variableregion that includes a variable framework region substantially from ahuman immunoglobulin or antibody and complementarity determining regions(CDRs) substantially from a non-human immunoglobulin or antibody.

The phrase “substantially from a human immunoglobulin or antibody” or“substantially human” means that, when aligned to a human immunoglobulinor antibody amino sequence for comparison purposes, the region shares atleast 80-90%, 90-95%, or 95-99% identity (i.e., local sequence identity)with the human framework or constant region sequence, allowing, forexample, for conservative substitutions, consensus sequencesubstitutions, germline substitutions, backmutations, and the like. Theintroduction of conservative substitutions, consensus sequencesubstitutions, germline substitutions, backmutations, and the like, isoften referred to as “optimization” of a humanized antibody or chain.The phrase “substantially from a non-human immunoglobulin or antibody”or “substantially non-human” means having an immunoglobulin or antibodysequence at least 80-95%, preferably at least 90-95%, more preferably,96%, 97%, 98%, or 99% identical to that of a non-human organism, e.g., anon-human mammal.

Accordingly, all regions or residues of a humanized immunoglobulin orantibody, or of a humanized immunoglobulin or antibody chain, exceptpossibly the CDRs, are substantially identical to the correspondingregions or residues of one or more native human immunoglobulinsequences. The term “corresponding region” or “corresponding residue”refers to a region or residue on a second amino acid or nucleotidesequence which occupies the same (i.e., equivalent) position as a regionor residue on a first amino acid or nucleotide sequence, when the firstand second sequences are optimally aligned for comparison purposes.

The term “significant identity” means that two polypeptide sequences,when optimally aligned, such as by the programs GAP or BESTFIT usingdefault gap weights, share at least 50-60% sequence identity, preferablyat least 60-70% sequence identity, more preferably at least 70-80%sequence identity, more preferably at least 80-90% identity, even morepreferably at least 90-95% identity, and even more preferably at least95% sequence identity or more (e.g., 99% sequence identity or more). Theterm “substantial identity” means that two polypeptide sequences, whenoptimally aligned, such as by the programs GAP or BESTFIT using defaultgap weights, share at least 80-90% sequence identity, preferably atleast 90-95% sequence identity, and more preferably at least 95%sequence identity or more (e.g., 99% sequence identity or more). Forsequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., Current Protocols in Molecular Biology). One example ofalgorithm that is suitable for determining percent sequence identity andsequence similarity is the BLAST algorithm, which is described inAltschul et al., J. Mol. Biol. 215:403 (1990). Software for performingBLAST analyses is publicly available through the National Center forBiotechnology Information (publicly accessible through the NationalInstitutes of Health NCBI internet server). Typically, default programparameters can be used to perform the sequence comparison, althoughcustomized parameters can also be used. For amino acid sequences, theBLASTP program uses as defaults a wordlength (W) of 3, an expectation(E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff,Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

Preferably, residue positions which are not identical differ byconservative amino acid substitutions. For purposes of classifying aminoacids substitutions as conservative or nonconservative, amino acids aregrouped as follows: Group I (hydrophobic sidechains): leu, met, ala,val, leu, ile; Group II (neutral hydrophilic side chains): cys, ser,thr; Group III (acidic side chains): asp, glu; Group IV (basic sidechains): asn, gln, his, lys, arg; Group V (residues influencing chainorientation): gly, pro; and Group VI (aromatic side chains): trp, tyr,phe. Conservative substitutions involve substitutions between aminoacids in the same class. Non-conservative substitutions constituteexchanging a member of one of these classes for a member of another.

Preferably, humanized immunoglobulins or antibodies bind antigen with anaffinity that is within a factor of three, four, or five of that of thecorresponding nonhumanized antibody. For example, if the nonhumanizedantibody has a binding affinity of 10⁹ M⁻¹, humanized antibodies willhave a binding affinity of at least 3×10⁹ M⁻¹, 4×10⁹ M⁻¹ or 5×10⁹ M⁻¹.When describing the binding properties of an immunoglobulin or antibodychain, the chain can be described based on its ability to “directantigen (e.g., Aβ) binding”. A chain is said to “direct antigen binding”when it confers upon an intact immunoglobulin or antibody (or antigenbinding fragment thereof) a specific binding property or bindingaffinity. A mutation (e.g., a backmutation) is said to substantiallyaffect the ability of a heavy or light chain to direct antigen bindingif it affects (e.g., decreases) the binding affinity of an intactimmunoglobulin or antibody (or antigen binding fragment thereof)comprising said chain by at least an order of magnitude compared to thatof the antibody (or antigen binding fragment thereof) comprising anequivalent chain lacking said mutation. A mutation “does notsubstantially affect (e.g., decrease) the ability of a chain to directantigen binding” if it affects (e.g., decreases) the binding affinity ofan intact immunoglobulin or antibody (or antigen binding fragmentthereof) comprising said chain by only a factor of two, three, or fourof that of the antibody (or antigen binding fragment thereof) comprisingan equivalent chain lacking said mutation.

The term “chimeric immunoglobulin” or antibody refers to animmunoglobulin or antibody whose variable regions derive from a firstspecies and whose constant regions derive from a second species.Chimeric immunoglobulins or antibodies can be constructed, for exampleby genetic engineering, from immunoglobulin gene segments belonging todifferent species. The terms “humanized immunoglobulin” or “humanizedantibody” are not intended to encompass chimeric immunoglobulins orantibodies, as defined infra. Although humanized immunoglobulins orantibodies are chimeric in their construction (i.e., comprise regionsfrom more than one species of protein), they include additional features(i.e., variable regions comprising donor CDR residues and acceptorframework residues) not found in chimeric immunoglobulins or antibodies,as defined herein.

An “antigen” is an entity (e.g., a proteinaceous entity or peptide) towhich an antibody specifically binds.

The term “epitope” or “antigenic determinant” refers to a site on anantigen to which an immunoglobulin or antibody (or antigen bindingfragment thereof) specifically binds. Epitopes can be formed both fromcontiguous amino acids or noncontiguous amino acids juxtaposed bytertiary folding of a protein. Epitopes formed from contiguous aminoacids are typically retained on exposure to denaturing solvents, whereasepitopes formed by tertiary folding are typically lost on treatment withdenaturing solvents. An epitope typically includes at least 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique spatialconformation. Methods of determining spatial conformation of epitopesinclude, for example, x-ray crystallography and 2-dimensional nuclearmagnetic resonance. See, e.g., Epitope Mapping Protocols in Methods inMolecular Biology, Vol. 66, G. E. Morris, Ed. (1996).

Antibodies that recognize the same epitope can be identified in a simpleimmunoassay showing the ability of one antibody to block the binding ofanother antibody to a target antigen, i.e., a competitive binding assay.Competitive binding is determined in an assay in which theimmunoglobulin under test inhibits specific binding of a referenceantibody to a common antigen, such as Aβ. Numerous types of competitivebinding assays are known, for example: solid phase direct or indirectradioimmunoassay (RIA), solid phase direct or indirect enzymeimmunoassay (EIA), sandwich competition assay (see Stahli et al.,Methods in Enzymology 9:242 (1983)); solid phase direct biotin-avidinEIA (see Kirkland et al., J. Immunol. 137:3614 (1986)); solid phasedirect labeled assay, solid phase direct labeled sandwich assay (seeHarlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborPress (1988)); solid phase direct label RIA using I-125 label (see Morelet al., Mol. Immunol. 25(1):7 (1988)); solid phase direct biotin-avidinEIA (Cheung et al., Virology 176:546 (1990)); and direct labeled RIA.(Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)). Typically, suchan assay involves the use of purified antigen bound to a solid surfaceor cells bearing either of these, an unlabeled test immunoglobulin and alabeled reference immunoglobulin. Competitive inhibition is measured bydetermining the amount of label bound to the solid surface or cells inthe presence of the test immunoglobulin. Usually the test immunoglobulinis present in excess. Usually, when a competing antibody is present inexcess, it will inhibit specific binding of a reference antibody to acommon antigen by at least 50-55%, 55-60%, 60-65%, 65-70% 70-75% ormore.

An epitope is also recognized by immunologic cells, for example, B cellsand/or T cells. Cellular recognition of an epitope can be determined byin vitro assays that measure antigen-dependent proliferation, asdetermined by ³H-thymidine incorporation, by cytokine secretion, byantibody secretion, or by antigen-dependent killing (cytotoxic Tlymphocyte assay).

Exemplary epitopes or antigenic determinants can be found within thehuman amyloid precursor protein (APP), but are preferably found withinthe Aβ peptide of APP. Multiple isoforms of APP exist, for exampleAPP⁶⁹⁵ APP⁷⁵¹ and APP⁷⁷⁰. Amino acids within APP are assigned numbersaccording to the sequence of the APP⁷⁷⁰ isoform (see e.g., GenBankAccession No. P05067). Aβ (also referred to herein as beta amyloidpeptide and A-beta) peptide is an approximately 4-kDa internal fragmentof 39-43 amino acids of APP (Aβ39, Aβ40, Aβ41, Aβ42 and Aβ43). Aβ40, forexample, consists of residues 672-711 of APP and Aβ42 consists ofresidues 673-713 of APP. As a result of proteolytic processing of APP bydifferent secretase enzymes iv vivo or in situ, Aβ is found in both a“short form”, 40 amino acids in length, and a “long form”, ranging from42-43 amino acids in length. Preferred epitopes or antigenicdeterminants, as described herein, are located within the N-terminus ofthe Aβ peptide and include residues within amino acids 1-10 of Aβ,preferably from residues 1-3, 1-4, 1-5, 1-6, 1-7 or 3-7 of Aβ42.Additional referred epitopes or antigenic determinants include residues2-4, 5, 6, 7 or 8 of Aβ, residues 3-5, 6, 7, 8 or 9 of Aβ, or residues4-7, 8, 9 or 10 of Aβ42. When an antibody is said to bind to an epitopewithin specified residues, such as Aβ 3-7, what is meant is that theantibody specifically binds to a polypeptide containing the specifiedresidues (i.e., Aβ 3-7 in this an example). Such an antibody does notnecessarily contact every residue within Aβ3-7. Nor does every singleamino acid substitution or deletion with in Aβ 3-7 necessarilysignificantly affect binding affinity.

The term “amyloidogenic disease” includes any disease associated with(or caused by) the formation or deposition of insoluble amyloid fibrils.Exemplary amyloidogenic diseases include, but are not limited tosystemic amyloidosis, Alzheimer's disease, mature onset diabetes,Parkinson's disease, Huntington's disease, fronto-temporal dementia, andthe prion-related transmissible spongiform encephalopathies (kuru andCreutzfeldt-Jacob disease in humans and scrapie and BSE in sheep andcattle, respectively). Different amyloidogenic diseases are defined orcharacterized by the nature of the polypeptide component of the fibrilsdeposited. For example, in subjects or patients having Alzheimer'sdisease, β-amyloid protein (e.g., wild-type, variant, or truncatedβ-amyloid protein) is the characterizing polypeptide component of theamyloid deposit. Accordingly, Alzheimer's disease is an example of a“disease characterized by deposits of Aβ” or a “disease associated withdeposits of Aβ”, e.g., in the brain of a subject or patient. The terms“β-amyloid protein”, “β-amyloid peptide”, “β-amyloid”, “Aβ” and “Aβpeptide” are used interchangeably herein.

An “immunogenic agent” or “immunogen” is capable of inducing animmunological response against itself on administration to a mammal,optionally in conjunction with an adjuvant.

The term “treatment” as used herein, is defined as the application oradministration of a therapeutic agent to a patient, or application oradministration of a therapeutic agent to an isolated tissue or cell linefrom a patient, who has a disease, a symptom of disease or apredisposition toward a disease, with the purpose to cure, heal,alleviate, relieve, alter, remedy, ameliorate, improve or affect thedisease, the symptoms of disease or the predisposition toward disease.

The term “effective dose” or “effective dosage” is defined as an amountsufficient to achieve or at least partially achieve the desired effect.The term “therapeutically effective dose” is defined as an amountsufficient to cure or at least partially arrest the disease and itscomplications in a patient already suffering from the disease. Amountseffective for this use will depend upon the severity of the infectionand the general state of the patient's own immune system.

The term “patient” includes human and other mammalian subjects thatreceive either prophylactic or therapeutic treatment.

“Soluble” or “dissociated” Aβ refers to non-aggregating or disaggregatedAβ polypeptide, including monomeric soluble as well as oligomericsoluble Aβ polypeptide (e.g., soluble Aβ dimers, trimers, and the like).“Insoluble” Aβ refers to aggregating Aβ polypeptide, for example, Aβheld together by noncovalent bonds. Aβ (e.g., Aβ42) is believed toaggregate, at least in part, due to the presence of hydrophobic residuesat the C-terminus of the peptide (part of the transmembrane domain ofAPP). Soluble Aβ can be found in vivo in biological fluids such ascerebrospinal fluid and/or serum. Alternatively, soluble Aβ can beprepared by dissolving lyophilized peptide in neat DMSO with sonication.The resulting solution is centrifuged (e.g., at 14,000×g, 4° C., 10minutes) to remove any insoluble particulates.

The term “effector function” refers to an activity that resides in theFc region of an antibody (e.g., an IgG antibody) and includes, forexample, the ability of the antibody to bind effector molecules such ascomplement and/or Fc receptors, which can control several immunefunctions of the antibody such as effector cell activity, lysis,complement-mediated activity, antibody clearance, and antibodyhalf-life.

The term “effector molecule” refers to a molecule that is capable ofbinding to the Fc region of an antibody (e.g., an IgG antibody)including, but not limited to, a complement protein or a Fc receptor.

The term “effector cell” refers to a cell capable of binding to the Fcportion of an antibody (e.g., an IgG antibody) typically via an Fcreceptor expressed on the surface of the effector cell including, butnot limited to, lymphocytes, e.g., antigen presenting cells and T cells.

The term “Fc region” refers to a C-terminal region of an IgG antibody,in particular, the C-terminal region of the heavy chain(s) of said IgGantibody. Although the boundaries of the Fc region of an IgG heavy chaincan vary slightly, a Fc region is typically defined as spanning fromabout amino acid residue Cys226 to the carboxyl-terminus of an IgG heavychain(s).

The term “Kabat numbering” unless otherwise stated, is defined as thenumbering of the residues in, e.g., an IgG heavy chain antibody usingthe EU index as in Kabat et al. (Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991)), expressly incorporated herein by reference.

The term “Fc receptor” or “FcR” refers to a receptor that binds to theFc region of an antibody. Typical Fc receptors which bind to an Fcregion of an antibody (e.g., an IgG antibody) include, but are notlimited to, receptors of the FcγRI, FcγRII, and FcγRIII subclasses,including allelic variants and alternatively spliced forms of thesereceptors. Fc receptors are reviewed in Ravetch and Kinet, Annu. Rev.Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); andde Haas et al., J. Lab. Clin. Med. 126:330-41 (1995).

I. Immunological and Therapeutic Reagents

Immunological and therapeutic reagents of the invention comprise orconsist of immunogens or antibodies, or functional or antigen bindingfragments thereof, as defined herein. The basic antibody structural unitis known to comprise a tetramer of subunits. Each tetramer is composedof two identical pairs of polypeptide chains, each pair having one“light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). Theamino-terminal portion of each chain includes a variable region of about100 to 110 or more amino acids primarily responsible for antigenrecognition. The carboxy-terminal portion of each chain defines aconstant region primarily responsible for effector function.

Light chains are classified as either kappa or lambda and are about 230residues in length. Heavy chains are classified as gamma (γ), mu (μ),alpha (α), delta (δ), or epsilon (ε), are about 450-600 residues inlength, and define the antibody's isotype as IgG, IgM, IgA, IgD and IgE,respectively. Both heavy and light chains are folded into domains. Theterm “domain” refers to a globular region of a protein, for example, animmunoglobulin or antibody. Immunoglobulin or antibody domains include,for example, three or four peptide loops stabilized by β-pleated sheetand an interchain disulfide bond. Intact light chains have, for example,two domains (V_(L) and C_(L)) and intact heavy chains have, for example,four or five domains (V_(H), C_(H)1, C_(H)2, and C_(H)3).

Within light and heavy chains, the variable and constant regions arejoined by a “J” region of about 12 or more amino acids, with the heavychain also including a “D” region of about 10 more amino acids. (Seegenerally, Fundamental Immunology (Paul, W., ed., 2nd ed. Raven Press,N.Y. (1989), Ch. 7, incorporated by reference in its entirety for allpurposes).

The variable regions of each light/heavy chain pair form the antibodybinding site. Thus, an intact antibody has two binding sites. Except inbifunctional or bispecific antibodies, the two binding sites are thesame. The chains all exhibit the same general structure of relativelyconserved framework regions (FR) joined by three hypervariable regions,also called complementarity determining regions or CDRs.Naturally-occurring chains or recombinantly produced chains can beexpressed with a leader sequence which is removed during cellularprocessing to produce a mature chain. Mature chains can also berecombinantly produced having a non-naturally occurring leader sequence,for example, to enhance secretion or alter the processing of aparticular chain of interest.

The CDRs of the two mature chains of each pair are aligned by theframework regions, enabling binding to a specific epitope. FromN-terminal to C-terminal, both light and heavy chains comprise thedomains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. “FR4” also is referredto in the art as the D/J region of the variable heavy chain and the Jregion of the variable light chain. The assignment of amino acids toeach domain is in accordance with the definitions of Kabat, Sequences ofProteins of Immunological Interest (National Institutes of Health,Bethesda, Md., 1987 and 1991). An alternative structural definition hasbeen proposed by Chothia et al., J. Mol. Biol. 196:901 (1987); Nature342:878 (1989); and J. Mol. Biol. 186:651 (1989) (hereinaftercollectively referred to as “Chothia et al.” and incorporated byreference in their entirety for all purposes).

A. Aβ Antibodies

Therapeutic agents of the invention include antibodies that specificallybind to Aβ or to other components of the amyloid plaque. Preferredantibodies are monoclonal antibodies. Some such antibodies bindspecifically to the aggregated form of Aβ without binding to the solubleform. Some bind specifically to the soluble form without binding to theaggregated form. Some bind to both aggregated and soluble forms.Antibodies used in therapeutic methods preferably have an intactconstant region or at least sufficient of the constant region tointeract with an Fc receptor. Preferred antibodies are those efficaciousat stimulating Fc-mediated phagocytosis of Aβ in plaques. Human isotypeIgG1 is preferred because of it having highest affinity of humanisotypes for the FcRI receptor on phagocytic cells (e.g., on brainresident macrophages or microglial cells). Human IgG1 is the equivalentof murine IgG2a, the latter thus suitable for testing in vivo efficacyin animal (e.g., mouse) models of Alzheimer's. Bispecific Fab fragmentscan also be used, in which one arm of the antibody has specificity forAβ, and the other for an Fc receptor. Preferred antibodies bind to Aβwith a binding affinity greater than (or equal to) about 10⁶, 10⁷, 10⁸,10⁹, or 10¹⁰ M⁻¹ (including affinities intermediate of these values).

Monoclonal antibodies bind to a specific epitope within Aβ that can be aconformational or nonconformational epitope. Prophylactic andtherapeutic efficacy of antibodies can be tested using the transgenicanimal model procedures described in the Examples. Preferred monoclonalantibodies bind to an epitope within residues 1-10 of Aβ (with the firstN terminal residue of natural Aβ designated 1), more preferably to anepitope within residues 3-7 of Aβ. In some methods, multiple monoclonalantibodies having binding specificities to different epitopes are used,for example, an antibody specific for an epitope within residues 3-7 ofAβ can be co-administered with an antibody specific for an epitopeoutside of residues 3-7 of Aβ. Such antibodies can be administeredsequentially or simultaneously. Antibodies to amyloid components otherthan Aβ can also be used (e.g., administered or co-administered).

Epitope specificity of an antibody can be determined, for example, byforming a phage display library in which different members displaydifferent subsequences of Aβ. The phage display library is then selectedfor members specifically binding to an antibody under test. A family ofsequences is isolated. Typically, such a family contains a common coresequence, and varying lengths of flanking sequences in differentmembers. The shortest core sequence showing specific binding to theantibody defines the epitope bound by the antibody. Antibodies can alsobe tested for epitope specificity in a competition assay with anantibody whose epitope specificity has already been determined. Forexample, antibodies that compete with the 12A11 antibody for binding toAβ bind to the same or similar epitope as 12A11, i.e., within residuesAβ 3-7. Screening antibodies for epitope specificity is a usefulpredictor of therapeutic efficacy. For example, an antibody determinedto bind to an epitope within residues 1-7 of Aβ is likely to beeffective in preventing and treating Alzheimer's disease according tothe methodologies of the present invention.

Antibodies that specifically bind to a preferred segment of Aβ withoutbinding to other regions of Aβ have a number of advantages relative tomonoclonal antibodies binding to other regions or polyclonal sera tointact Aβ. First, for equal mass dosages, dosages of antibodies thatspecifically bind to preferred segments contain a higher molar dosage ofantibodies effective in clearing amyloid plaques. Second, antibodiesspecifically binding to preferred segments can induce a clearingresponse against amyloid deposits without inducing a clearing responseagainst intact APP polypeptide, thereby reducing the potential sideeffects.

1. Production of Nonhuman Antibodies

The present invention features non-human antibodies, for example,antibodies having specificity for the preferred Aβ epitopes of theinvention. Such antibodies can be used in formulating varioustherapeutic compositions of the invention or, preferably, providecomplementarity determining regions for the production of humanized orchimeric antibodies (described in detail below). The production ofnon-human monoclonal antibodies, e.g., murine, guinea pig, primate,rabbit or rat, can be accomplished by, for example, immunizing theanimal with Aβ. A longer polypeptide comprising Aβ or an immunogenicfragment of Aβ or anti-idiotypic antibodies to an antibody to Aβ canalso be used. See Harlow & Lane, supra, incorporated by reference forall purposes). Such an immunogen can be obtained from a natural source,by peptide synthesis or by recombinant expression. Optionally, theimmunogen can be administered fused or otherwise complexed with acarrier protein, as described below. Optionally, the immunogen can beadministered with an adjuvant. The term “adjuvant” refers to a compoundthat when administered in conjunction with an antigen augments theimmune response to the antigen, but when administered alone does notgenerate an immune response to the antigen. Adjuvants can augment animmune response by several mechanisms including lymphocyte recruitment,stimulation of B and/or T cells, and stimulation of macrophages. Severaltypes of adjuvant can be used as described below. Complete Freund'sadjuvant followed by incomplete adjuvant is preferred for immunizationof laboratory animals.

Rabbits or guinea pigs are typically used for making polyclonalantibodies. Exemplary preparation of polyclonal antibodies, e.g., forpassive protection, can be performed as follows. 125 non-transgenic miceare immunized with 100 μg Aβ 1-42, plus CFA/IFA adjuvant, and euthanizedat 4-5 months. Blood is collected from immunized mice. IgG is separatedfrom other blood components. Antibody specific for the immunogen may bepartially purified by affinity chromatography. An average of about 0.5-1mg of immunogen-specific antibody is obtained per mouse, giving a totalof 60-120 mg.

Mice are typically used for making monoclonal antibodies. Monoclonalscan be prepared against a fragment by injecting the fragment or longerform of Aβ into a mouse, preparing hybridomas and screening thehybridomas for an antibody that specifically binds to Aβ. Optionally,antibodies are screened for binding to a specific region or desiredfragment of Aβ without binding to other nonoverlapping fragments of Aβ.The latter screening can be accomplished by determining binding of anantibody to a collection of deletion mutants of an Aβ peptide anddetermining which deletion mutants bind to the antibody. Binding can beassessed, for example, by Western blot or ELISA. The smallest fragmentto show specific binding to the antibody defines the epitope of theantibody. Alternatively, epitope specificity can be determined by acompetition assay is which a test and reference antibody compete forbinding to Aβ. If the test and reference antibodies compete, then theybind to the same epitope or epitopes sufficiently proximal such thatbinding of one antibody interferes with binding of the other. Thepreferred isotype for such antibodies is mouse isotype IgG2a orequivalent isotype in other species. Mouse isotype IgG2a is theequivalent of human isotype IgG1 (e.g., human IgG1).

2. Chimeric and Humanized Antibodies

The present invention also features chimeric and/or humanized antibodies(i.e., chimeric and/or humanized immunoglobulins) specific for betaamyloid peptide. Chimeric and/or humanized antibodies have the same orsimilar binding specificity and affinity as a mouse or other nonhumanantibody that provides the starting material for construction of achimeric or humanized antibody.

a. Production of Chimeric Antibodies

The term “chimeric antibody” refers to an antibody whose light and heavychain genes have been constructed, typically by genetic engineering,from immunoglobulin gene segments belonging to different species. Forexample, the variable (V) segments of the genes from a mouse monoclonalantibody may be joined to human constant (C) segments, such as IgG1 andIgG4. Human isotype IgG1 is preferred. A typical chimeric antibody isthus a hybrid protein consisting of the V or antigen-binding domain froma mouse antibody and the C or effector domain from a human antibody.

b. Production of Humanized Antibodies

The term “humanized antibody” refers to an antibody comprising at leastone chain comprising variable region framework residues substantiallyfrom a human antibody chain (referred to as the acceptor immunoglobulinor antibody) and at least one complementarity determining regionsubstantially from a mouse antibody, (referred to as the donorimmunoglobulin or antibody). See, Queen et al., Proc. Natl. Acad. Sci.USA 86:10029-10033 (1989), U.S. Pat. No. 5,530,101, U.S. Pat. No.5,585,089, U.S. Pat. No. 5,693,761, U.S. Pat. No. 5,693,762, Selick etal., WO 90/07861, and Winter, U.S. Pat. No. 5,225,539 (incorporated byreference in their entirety for all purposes). The constant region(s),if present, are also substantially or entirely from a humanimmunoglobulin.

The substitution of mouse CDRs into a human variable domain framework ismost likely to result in retention of their correct spatial orientationif the human variable domain framework adopts the same or similarconformation to the mouse variable framework from which the CDRsoriginated. This is achieved by obtaining the human variable domainsfrom human antibodies whose framework sequences exhibit a high degree ofsequence identity with the murine variable framework domains from whichthe CDRs were derived. The heavy and light chain variable frameworkregions can be derived from the same or different human antibodysequences. The human antibody sequences can be the sequences ofnaturally occurring human antibodies or can be consensus sequences ofseveral human antibodies. See Kettleborough et al., Protein Engineering4:773 (1991); Kolbinger et al., Protein Engineering 6:971 (1993) andCarter et al., WO 92/22653.

Having identified the complementarity determining regions of the murinedonor immunoglobulin and appropriate human acceptor immunoglobulins, thenext step is to determine which, if any, residues from these componentsshould be substituted to optimize the properties of the resultinghumanized antibody. In general, substitution of human amino acidresidues with murine should be minimized, because introduction of murineresidues increases the risk of the antibody eliciting ahuman-anti-mouse-antibody (HAMA) response in humans. Art-recognizedmethods of determining immune response can be performed to monitor aHAMA response in a particular patient or during clinical trials.Patients administered humanized antibodies can be given animmunogenicity assessment at the beginning and throughout theadministration of said therapy. The HAMA response is measured, forexample, by detecting antibodies to the humanized therapeutic reagent,in serum samples from the patient using a method known to one in theart, including surface plasmon resonance technology (BIACORE) and/orsolid-phase ELISA analysis.

Certain amino acids from the human variable region framework residuesare selected for substitution based on their possible influence on CDRconformation and/or binding to antigen. The unnatural juxtaposition ofmurine CDR regions with human variable framework region can result inunnatural conformational restraints, which, unless corrected bysubstitution of certain amino acid residues, lead to loss of bindingaffinity.

The selection of amino acid residues for substitution is determined, inpart, by computer modeling. Computer hardware and software are describedherein for producing three-dimensional images of immunoglobulinmolecules. In general, molecular models are produced starting fromsolved structures for immunoglobulin chains or domains thereof. Thechains to be modeled are compared for amino acid sequence similaritywith chains or domains of solved three-dimensional structures, and thechains or domains showing the greatest sequence similarity is/areselected as starting points for construction of the molecular model.Chains or domains sharing at least 50% sequence identity are selectedfor modeling, and preferably those sharing at least 60%, 70%, 80%, 90%sequence identity or more are selected for modeling. The solved startingstructures are modified to allow for differences between the actualamino acids in the immunoglobulin chains or domains being modeled, andthose in the starting structure. The modified structures are thenassembled into a composite immunoglobulin. Finally, the model is refinedby energy minimization and by verifying that all atoms are withinappropriate distances from one another and that bond lengths and anglesare within chemically acceptable limits.

The selection of amino acid residues for substitution can also bedetermined, in part, by examination of the characteristics of the aminoacids at particular locations, or empirical observation of the effectsof substitution or mutagenesis of particular amino acids. For example,when an amino acid differs between a murine variable region frameworkresidue and a selected human variable region framework residue, thehuman framework amino acid should usually be substituted by theequivalent framework amino acid from the mouse antibody when it isreasonably expected that the amino acid:

-   -   (1) noncovalently binds antigen directly,    -   (2) is adjacent to a CDR region,    -   (3) otherwise interacts with a CDR region (e.g., is within about        3-6 Å of a CDR region as determined by computer modeling), or    -   (4) participates in the VL-VH interface.

Residues which “noncovalently bind antigen directly” include amino acidsin positions in framework regions which have a good probability ofdirectly interacting with amino acids on the antigen according toestablished chemical forces, for example, by hydrogen bonding, Van derWaals forces, hydrophobic interactions, and the like.

CDR and framework regions are as defined by Kabat et al. or Chothia etal., supra. When framework residues, as defined by Kabat et al., supra,constitute structural loop residues as defined by Chothia et al., supra,the amino acids present in the mouse antibody may be selected forsubstitution into the humanized antibody. Residues which are “adjacentto a CDR region” include amino acid residues in positions immediatelyadjacent to one or more of the CDRs in the primary sequence of thehumanized immunoglobulin chain, for example, in positions immediatelyadjacent to a CDR as defined by Kabat, or a CDR as defined by Chothia(See e.g., Chothia and Lesk JMB 196:901 (1987)). These amino acids areparticularly likely to interact with the amino acids in the CDRs and, ifchosen from the acceptor, to distort the donor CDRs and reduce affinity.Moreover, the adjacent amino acids may interact directly with theantigen (Amit et al., Science, 233:747 (1986), which is incorporatedherein by reference) and selecting these amino acids from the donor maybe desirable to keep all the antigen contacts that provide affinity inthe original antibody.

Residues that “otherwise interact with a CDR region” include those thatare determined by secondary structural analysis to be in a spatialorientation sufficient to affect a CDR region. In one embodiment,residues that “otherwise interact with a CDR region” are identified byanalyzing a three-dimensional model of the donor immunoglobulin (e.g., acomputer-generated model). A three-dimensional model, typically of theoriginal donor antibody, shows that certain amino acids outside of theCDRs are close to the CDRs and have a good probability of interactingwith amino acids in the CDRs by hydrogen bonding, Van der Waals forces,hydrophobic interactions, etc. At those amino acid positions, the donorimmunoglobulin amino acid rather than the acceptor immunoglobulin aminoacid may be selected. Amino acids according to this criterion willgenerally have a side chain atom within about 3 angstrom units (Å) ofsome atom in the CDRs and must contain an atom that could interact withthe CDR atoms according to established chemical forces, such as thoselisted above.

In the case of atoms that may form a hydrogen bond, the 3 Å is measuredbetween their nuclei, but for atoms that do not form a bond, the 3 Å ismeasured between their Van der Waals surfaces. Hence, in the lattercase, the nuclei must be within about 6 Å (3 Å plus the sum of the Vander Waals radii) for the atoms to be considered capable of interacting.In many cases the nuclei will be from 4 or 5 to 6 Å apart. Indetermining whether an amino acid can interact with the CDRs, it ispreferred not to consider the last 8 amino acids of heavy chain CDR 2 aspart of the CDRs, because from the viewpoint of structure, these 8 aminoacids behave more as part of the framework.

Amino acids that are capable of interacting with amino acids in theCDRs, may be identified in yet another way. The solvent accessiblesurface area of each framework amino acid is calculated in two ways: (1)in the intact antibody, and (2) in a hypothetical molecule consisting ofthe antibody with its CDRs removed. A significant difference betweenthese numbers of about 10 square angstroms or more shows that access ofthe framework amino acid to solvent is at least partly blocked by theCDRs, and therefore that the amino acid is making contact with the CDRs.Solvent accessible surface area of an amino acid may be calculated basedon a three-dimensional model of an antibody, using algorithms known inthe art (e.g., Connolly, J. Appl. Cryst. 16:548 (1983) and Lee andRichards, J. Mol. Biol. 55:379 (1971), both of which are incorporatedherein by reference). Framework amino acids may also occasionallyinteract with the CDRs indirectly, by affecting the conformation ofanother framework amino acid that in turn contacts the CDRs.

The amino acids at several positions in the framework are known to beimportant for determining CDR confirmation (e.g., capable of interactingwith the CDRs) in many antibodies (Chothia and Lesk, supra, Chothia etal., supra and Tramontano et al., J. Mol. Biol. 215:175 (1990), all ofwhich are incorporated herein by reference). These authors identifiedconserved framework residues important for CDR conformation by analysisof the structures of several known antibodies. The antibodies analyzedfell into a limited number of structural or “canonical” classes based onthe conformation of the CDRs. Conserved framework residues withinmembers of a canonical class are referred to as “canonical” residues.Canonical residues include residues 2, 25, 29, 30, 33, 48, 64, 71, 90,94 and 95 of the light chain and residues 24, 26, 29, 34, 54, 55, 71 and94 of the heavy chain. Additional residues (e.g., CDR structuredetermining residues) can be identified according to the methodology ofMartin and Thorton (1996) J. Mol. Biol. 263:800. Notably, the aminoacids at positions 2, 48, 64 and 71 of the light chain and 26-30, 71 and94 of the heavy chain (numbering according to Kabat) are known to becapable of interacting with the CDRs in many antibodies. The amino acidsat positions 35 in the light chain and 93 and 103 in the heavy chain arealso likely to interact with the CDRs. Additional residues which mayeffect conformation of the CDRs can be identified according to themethodology of Foote and Winter (1992) J. Mol. Biol. 224:487. Suchresidues are termed “vernier” residues and are those residues in theframework region closely underlying (i.e., forming a “platform” tinder)the CDRs. At all these numbered positions, choice of the donor aminoacid rather than the acceptor amino acid (when they differ) to be in thehumanized immunoglobulin is preferred. On the other hand, certainresidues capable of interacting with the CDR region, such as the first 5amino acids of the light chain, may sometimes be chosen from theacceptor immunoglobulin without loss of affinity in the humanizedimmunoglobulin.

Residues which “participate in the VL-VH interface” or “packingresidues” include those residues at the interface between VL and VH asdefined, for example, by Novotny and Haber, Proc. Natl. Acad. Sci. USA,82:4592-66 (1985) or Chothia et al, supra. Generally, unusual packingresidues should be retained in the humanized antibody if they differfrom those in the human frameworks.

In general, one or more of the amino acids fulfilling the above criteriacan be substituted. In some embodiments, all or most of the amino acidsfulfilling the above criteria are substituted. Occasionally, there issome ambiguity about whether a particular amino acid meets the abovecriteria, and alternative variant immunoglobulins are produced, one ofwhich has that particular substitution, the other of which does not.Alternative variant immunoglobulins so produced can be tested in any ofthe assays described herein for the desired activity, and the preferredimmunoglobulin selected.

Usually the CDR regions in humanized antibodies are substantiallyidentical, and more usually, identical to the corresponding CDR regionsof the donor antibody. However, in certain embodiments, it may bedesirable to modify one or more CDR regions to modify the antigenbinding specificity of the antibody and/or reduce the immunogenicity ofthe antibody. Typically, one or more residues of a CDR are altered tomodify binding to achieve a more favored on-rate of binding, a morefavored off-rate of binding, or both, such that an idealized bindingconstant is achieved. Using this strategy, an antibody having ultra highbinding affinity of, for example, 10¹⁰ M⁻¹ or more, can be achieved.Briefly, the donor CDR sequence is referred to as a base sequence fromwhich one or more residues are then altered. Affinity maturationtechniques, as described herein, can be used to alter the CDR region(s)followed by screening of the resultant binding molecules for the desiredchange in binding. The method may also be used to alter the donor CDR,typically a mouse CDR, to be less immunogenic such that a potentialhuman anti-mouse antibody (HAMA) response is minimized or avoided.Accordingly, as CDR(s) are altered, changes in binding affinity as wellas immunogenicity can be monitored and scored such that an antibodyoptimized for the best combined binding and low immunogenicity areachieved (see, e.g., U.S. Pat. No. 6,656,467 and U.S. Pat. Pub.US20020164326A1).

In another approach, the CDR regions of the antibody are analyzed todetermine the contributions of each individual CDR to antibody bindingand/or immunogenicity by systemically replacing each of the donor CDRswith a human counterpart. The resultant panel of humanized antibodies isthen scored for antigen affinity and potential immunogenicity of eachCDR. In this way, the two clinically important properties of a candidatebinding molecule, i.e., antigen binding and low immunogenicity, aredetermined. If patient sera against a corresponding murine orCDR-grafted (humanized) form of the antibody is available, then theentire panel of antibodies representing the systematic human CDRexchanges can be screened to determine the patients anti-idiotypicresponse against each donor CDR (for technical details, see, e.g.,Iwashi et al., Mol. Immunol. 36:1079-91 (1999). Such an approach allowsfor identifying essential donor CDR regions from non-essential donorCDRs. Nonessential donor CDR regions may then be exchanged with a humancounterpart CDR. Where an essential CDR region cannot be exchangedwithout unacceptable loss of function, identification of thespecificity-determining residues (SDRs) of the CDR is performed by, forexample, site-directed mutagenesis. In this way, the CDR can then bereengineered to retain only the SDRs and be human and/or minimallyimmunogenic at the remaining amino acid positions throughout the CDR.Such an approach, where only a portion of the donor CDR is grafted, isalso referred to as abbreviated CDR-grafting (for technical details onthe foregoing techniques, see, e.g., Tamura et al., J. of Immunology164(3): 1432-41. (2000); Gonzales et al., Mol. Immunol 40:337-349(2003); Kashmiri et al., Crit. Rev. Oncol. Hematol. 38:3-16 (2001); andDe Pascalis et al., J. of Immunology 169(6):3076-84. (2002).

Moreover, it is sometimes possible to make one or more conservativeamino acid substitutions of CDR residues without appreciably affectingthe binding affinity of the resulting humanized immunoglobulin. Byconservative substitutions are intended combinations such as gly, ala;val, ile, leu; asp, glu; asn, gln; ser, thr; lys, arg; and phe, tyr.

Additional candidates for substitution are acceptor human frameworkamino acids that are unusual or “rare” for a human immunoglobulin atthat position. These amino acids can be substituted with amino acidsfrom the equivalent position of the mouse donor antibody or from theequivalent positions of more typical human immunoglobulins. For example,substitution may be desirable when the amino acid in a human frameworkregion of the acceptor immunoglobulin is rare for that position and thecorresponding amino acid in the donor immunoglobulin is common for thatposition in human immunoglobulin sequences; or when the amino acid inthe acceptor immunoglobulin is rare for that position and thecorresponding amino acid in the donor immunoglobulin is also rare,relative to other human sequences. Whether a residue is rare foracceptor human framework sequences should also be considered whenselecting residues for backmutation based on contribution to CDRconformation. For example, if backmutation results in substitution of aresidue that is rare for acceptor human framework sequences, a humanizedantibody may be tested with and without for activity. If thebackmutation is not necessary for activity, it may be eliminated toreduce immunogenicity concerns. For example, backmutation at thefollowing residues may introduce a residue that is rare in acceptorhuman framework sequences; uk=v2(2.0%), L3 (0.4%), T7 (1.8%), Q18(0.2%), L83 (1.2%), I85 (2.9%), A100 (0.3%) and L106 (1.1%); and vh=T3(2.0%), KS (1.8%), I11 (0.2%), S23 (1.5%), F24 (1.5%), S41 (2.3%), K71(2.4%), R75 (1.4%), I82 (1.4%), D83 (2.2%) and L109 (0.8%). Thesecriteria help ensure that an atypical amino acid in the human frameworkdoes not disrupt the antibody structure. Moreover, by replacing anunusual human acceptor amino acid with an amino acid from the donorantibody that happens to be typical for human antibodies, the humanizedantibody may be made less immunogenic.

The term “rare”, as used herein, indicates an amino acid occurring atthat position in less than about 20%, preferably less than about 10%,more preferably less than about 5%, even more preferably less than about3%, even more preferably less than about 2% and even more preferablyless than about 1% of sequences in a representative sample of sequences,and the term “common”, as used herein, indicates an amino acid occurringin more than about 25% but usually more than about 50% of sequences in arepresentative sample. For example, when deciding whether an amino acidin a human acceptor sequence is “rare” or “common”, it will often bepreferable to consider only human variable region sequences and whendeciding whether a mouse amino acid is “rare” or “common”, only mousevariable region sequences. Moreover, all human light and heavy chainvariable region sequences are respectively grouped into “subgroups” ofsequences that are especially homologous to each other and have the sameamino acids at certain critical positions (Kabat et al., supra). Whendeciding whether an amino acid in a human acceptor sequence is “rare” or“common” among human sequences, it will often be preferable to consideronly those human sequences in the same subgroup as the acceptorsequence.

Additional candidates for substitution are acceptor human frameworkamino acids that would be identified as part of a CDR region under thealternative definition proposed by Chothia et al., supra. Additionalcandidates for substitution are acceptor human framework amino acidsthat would be identified as part of a CDR region under the AbM and/orcontact definitions.

Additional candidates for substitution are acceptor framework residuesthat correspond to a rare or unusual donor framework residue. Rare orunusual donor framework residues are those that are rare or unusual (asdefined herein) for murine antibodies at that position. For murineantibodies, the subgroup can be determined according to Kabat andresidue positions identified which differ from the consensus. Thesedonor specific differences may point to somatic mutations in the murinesequence which enhance activity. Unusual residues that are predicted toaffect binding (e.g., packing canonical and/or vernier residues) areretained, whereas residues predicted to be unimportant for binding canbe substituted. Rare residues within the 12A11 UK sequence include I85(3.6%). Rare residues within the 12A11 vh sequence include T3 (1.0%),I11(1.7%), L12 (1.7%), S41 (2.8%), D83 (1.8%) and A85 (1.8%).

Additional candidates for substitution are non-germline residuesoccurring in an acceptor framework region. For example, when an acceptorantibody chain (i.e., a human antibody chain sharing significantsequence identity with the donor antibody chain) is aligned to agermline antibody chain (likewise sharing significant sequence identitywith the donor chain), residues not matching between acceptor chainframework and the germline chain framework can be substituted withcorresponding residues from the germline sequence.

Other than the specific amino acid substitutions discussed above, theframework regions of humanized immunoglobulins are usually substantiallyidentical, and more usually, identical to the framework regions of thehuman antibodies from which they were derived. Of course, many of theamino acids in the framework region make little or no directcontribution to the specificity or affinity of an antibody. Thus, manyindividual conservative substitutions of framework residues can betolerated without appreciable change of the specificity or affinity ofthe resulting humanized immunoglobulin. Thus, in one embodiment thevariable framework region of the humanized immunoglobulin shares atleast 85% sequence identity to a human variable framework regionsequence or consensus of such sequences. In another embodiment, thevariable framework region of the humanized immunoglobulin shares atleast 90%, preferably 95%, more preferably 96%, 97%, 98% or 99% sequenceidentity to a human variable framework region sequence or consensus ofsuch sequences. In general, however, such substitutions are undesirable.

In exemplary embodiments, the humanized antibodies of the inventionexhibit a specific binding affinity for antigen of at least 10⁷, 10⁸,10⁹ or 10¹⁰M⁻¹. In other embodiments, the antibodies of the inventioncan have binding affinities of at least 10¹⁰, 10¹¹ or 10¹² M⁻¹. Usuallythe upper limit of binding affinity of the humanized antibodies forantigen is within a factor of three, four or five of that of the donorimmunoglobulin. Often the lower limit of binding affinity is also withina factor of three, four or five of that of donor immunoglobulin.Alternatively, the binding affinity can be compared to that of ahumanized antibody having no substitutions (e.g., an antibody havingdonor CDRs and acceptor FRs, but no FR substitutions). In suchinstances, the binding of the optimized antibody (with substitutions) ispreferably at least two- to three-fold greater, or three- to four-foldgreater, than that of the unsubstituted antibody. For makingcomparisons, activity of the various antibodies can be determined, forexample, by BIACORE (i.e., surface plasmon resonance using unlabelledreagents) or competitive binding assays.

c. Production of Humanized 12A11 Antibodies

A preferred embodiment of the present invention features a humanizedantibody to the N-terminus of Aβ, in particular, for use in thetherapeutic and/or diagnostic methodologies described herein. Aparticularly preferred starting material for production of humanizedantibodies is the monoclonal antibody 12A11. 12A11 is specific for theN-terminus of Aβ and has been shown to (1) have a high avidity foraggregated Aβ1-42, (2) have the ability to capture soluble Aβ, and (3)mediate phagocytosis (e.g., induce phagocytosis) of amyloid plaque (seeExample I). The in vivo efficacy of the 12A11 antibody is described inExample II. The cloning and sequencing of cDNA encoding the 12A11antibody heavy and light chain variable regions is described in ExampleIII.

Suitable human acceptor antibody sequences can be identified by computercomparisons of the amino acid sequences of the mouse variable regionswith the sequences of known human antibodies. The comparison isperformed separately for heavy and light chains but the principles aresimilar for each. In particular, variable domains from human antibodieswhose framework sequences exhibit a high degree of sequence identitywith the murine VL and VH framework regions are identified by query of,for example, the Kabat Database or the IgG Protein Sequence Databaseusing NCBI IgG BLAST (publicly accessible through the NationalInstitutes of Health NCBI internet server) with the respective murineframework sequences. In one embodiment, acceptor sequences sharinggreater that 50% sequence identity with murine donor sequences, e.g.,donor framework (FR) sequences, are selected. Preferably, acceptorantibody sequences sharing 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%sequence identity or more are selected.

A computer comparison of 12A11 revealed that the 12A11 light chain(mouse subgroup II) shows the greatest sequence identity to human lightchains of subtype kappa II, and that the 12A11 heavy chain (mousesubgroup Ib) shows greatest sequence identity to human heavy chains ofsubtype II, as defined by Kabat et al., supra. Light and heavy humanframework regions can be derived from human antibodies of thesesubtypes, or from consensus sequences of such subtypes. In a firsthumanization effort, light chain variable framework regions were derivedfrom human subgroup II antibodies. Based on previous experimentsdesigned to achieve high levels of expression of humanized antibodieshaving heavy chain variable framework regions derived from humansubgroup II antibodies, it had been discovered that expression levels ofsuch antibodies were sometimes low. Accordingly, based on the reasoningdescribed in Saldanha et al. (1999) Mol Immunol. 36:709-19, frameworkregions from human subgroup III antibodies were chosen rather than humansubgroup II.

A human subgroup II antibody K64(AIMS4) (accession no. BAC01733) wasidentified from the NCBI non-redundant database having significantsequence identity within the light chain variable regions of 12A11. Ahuman subgroup III antibody M72 (accession no. AAA69734) was identifiedfrom the NCBI non-redundant database having significant sequenceidentity within the heavy chain variable regions of 12A11 (see alsoSchroeder and Wang (1990) Proc. Natl. Acad. Sci. U.S.A. 872: 6146-6150.

Alternative light chain acceptor sequences include, for example, PDBAccession No. 1KFA (gi24158782), PDB Accession No. 1KFA (gi24158784),EMBL Accession No. CAE75574.1 (gi38522587), EMBL Accession No.CAE75575.1 (gi38522590), EMBL Accession No. CAE84952.1 (gi39838891), DJBAccession No. BAC01734.1 (gi21669419), DJB Accession No. BAC01730.1(gi21669411), PIR Accession No. S40312 (gi481978), EMBL Accession No.CAA51090.1 (gi3980118), GenBank Accession No. AAH63599.1 (gi39794308),PIR Accession No. S22902 (gil 06540), PIR Accession No. S42611(gi631215), EMBL Accession No. CAA38072.1 (gi433890), GenBank AccessionNo. AAD00856.1 (gi4100384), EMBL Accession No. CAA39072.1 (gi34000), PIRAccession No. S23230 (gi284256), DBJ Accession No. BAC01599.1(gi21669149), DBJ Accession No. BAC01729.1 (gi21669409), DBJ AccessionNo. BAC01562.1 (gi21669075), EMBL Accession No. CAA85590.1 (gi587338),GenBank Accession No. AAQ99243.1 (gi37694665), GenBank Accession No.AAK94811.1 (gi18025604), EMBL Accession No. CAB51297.1 (gi5578794), DBJAccession No. BAC01740.1 (gi21669431), and DBJ Accession No. BAC01733.1(gi21669417). Alternative heavy chain acceptor sequences include, forexample, GenBank Accession No. AAB35009.1 (gi1041885), DBJ Accession No.BAC01904.1 (gi21669789), GenBank Accession No. AAD53816.1 (gi5834100),GenBank Accession No. AAS86081.1 (gi46254223), DBJ Accession No.BAC01462.1 (gi21668870), GenBank Accession No. AAC18191.1 (gi3170773),DBJ Accession No. BAC02266.1 (gi21670513), GenBank Accession No.AAD56254.1 (gi5921589), GenBank Accession No. AAD53807.1 (gi5834082),DBJ Accession No. BAC02260.1 (gi21670501), GenBank Accession No.AAC18166.1 (gi3170723), EMBL Accession No. CAA49495.1 (gi33085), PIRAccession No. S31513 (gi345903), GenBank Accession No. AAS86079.1(gi46254219), DBJ Accession No. BAC01917.1 (gi21669815), DBJ AccessionNo. BAC01912.1 (gi21669805), GenBank Accession No. AAC18283.1(gi3170961), DBJ Accession No. BAC01903 (gi21669787), DBJ Accession NO.BAC01887.1 (gi21669755), DBJ Accession No. BAC02259.1 (gi21370499), DBJAccession No. BAC01913.1 (gi21669807), DBJ Accession No. BAC01910.1(gi21669801), DJB Accession No. BAC02267.1 (gi21670515), GenBankAccession No. AAC18306.1 (gi3171011), GenBank Accession No. AAD53817.1(gi5834102), PIR Accession No. E36005 (gi106423), EMBL CAB37129.1(gi4456494) and GenBank AAA68892.1 (gi186190).

In exemplary embodiments, humanized antibodies of the invention include12A11 CDRs and FRs from an acceptor sequence listed supra. Residueswithin the framework regions important for CDR conformation and/oractivity as described herein are selected for backmutation (if differingbetween donor and acceptor sequences).

Residues are next selected for substitution, as follows. When an aminoacid differs between a 12A11 variable framework region and an equivalenthuman variable framework region, the human framework amino acid shouldusually be substituted by the equivalent mouse amino acid if it isreasonably expected that the amino acid:

-   -   (1) noncovalently binds antigen directly,    -   (2) is adjacent to a CDR region, is part of a CDR region under        the alternative definition proposed by Chothia et al., supra, or        otherwise interacts with a CDR region (e.g., is within about 3A        of a CDR region), or    -   (3) participates in the VL-VH interface.

Structural analysis of the 12A11 antibody heavy and light chain variableregions, and humanization of the 12A11 antibody is described in ExampleV. Briefly, three-dimensional models for the solved murine antibodystructures 1 KTR for the light chain and 1JRH and 1ETZ for the heavychain were studied. Alternative three-dimensional models which can bestudied for identification of residues, important for CDR confirmation(e.g., vernier residues), include PDB Accession No. 2JEL (gi3212688),PDB Accession No. 1TET (gi494639), PDB Accession No. IJP5 (gi16975307),PDB Accession No. 1CBV (gi493917), PDB Accession No. 2PCP (gi4388943),PDB Accession No. 1191 (gi2050118), PDB Accession No. 1CLZ (gi1827926),PDB Accession No. 1FL6 (gi17942615) and PDB Accession No. 1KEL (gi1942968) for the light chain and PDB 1GGI (gi442938), PDB Accession No.1GGB (gi442934), PDB Accession No. 1N5Y (gi28373913), PDB Accession No.2HMI (gi3891821), PDB Accession No. 1FDL (gi229915), PDB Accession No.1KIP (gi1942788), PDB Accession No. 1KIQ (gi1942791) and PDB AccessionNo. 1VFA (gi576325) for the heavy chain.

Three-dimensional structural information for the antibodies describedherein is publicly available, for example, from the ResearchCollaboratory for Structural Bioinformatics' Protein Data Bank (PDB).The PDB is freely accessible via the World Wide Web internet and isdescribed by Berman et al. (2000) Nucleic Acids Research, 28:235. Studyof solved three-dimensional structures allows for the identification ofCDR-interacting residues within 12A11. Alternatively, three-dimensionalmodels for the 12A11 VH and VL chains can be generated using computermodeling software. Briefly, a three-dimensional model is generated basedon the closest solved murine antibody structures for the heavy and lightchains. For this purpose, 1KTR can be used as a template for modelingthe 12A11 light chain, and 1ETZ and 1JRH used as templates for modelingthe heavy chain. The model can be further refined by a series of energyminimization steps to relieve unfavorable atomic contacts and optimizeelectrostatic and van der Waals interactions. Additionalthree-dimensional analysis and/or modeling can be performed using 2JEL(2.5 Å) and/or 1TET (2.3 Å) for the light chain and 1GGI (2.8 Å) for theheavy chain (or other antibodies set forth supra) based on thesimilarity between these solved murine structures and the respective12A11 chains.

The computer model of the structure of 12A11 can further serve as astarting point for predicting the three-dimensional structure of anantibody containing the 12A11 complementarity determining regionssubstituted in human framework structures. Additional models can beconstructed representing the structure as further amino acidsubstitutions are introduced.

In general, substitution of one, most or all of the amino acidsfulfilling the above criteria is desirable. Accordingly, the humanizedantibodies of the present invention will usually contain a substitutionof a human light chain framework residue with a corresponding 12A11residue in at least 1, 2, 3 or more of the chosen positions. Thehumanized antibodies also usually contain a substitution of a humanheavy chain framework residue with a corresponding 12A11 residue in atleast 1, 2, 3 or more of the chosen positions.

Occasionally, however, there is some ambiguity about whether aparticular amino acid meets the above criteria, and alternative variantimmunoglobulins are produced, one of which has that particularsubstitution, the other of which does not. In instances wheresubstitution with a murine residue would introduce a residue that israre in human immunoglobulins at a particular position, it may bedesirable to test the antibody for activity with or without theparticular substitution. If activity (e.g., binding affinity and/orbinding specificity) is about the same with or without the substitution,the antibody without substitution may be preferred, as it would beexpected to elicit less of a HAMA response, as described herein.

Other candidates for substitution are acceptor human framework aminoacids that are unusual for a human immunoglobulin at that position.These amino acids can be substituted with amino acids from theequivalent position of more typical human immunoglobulins.Alternatively, amino acids from equivalent positions in the mouse 12A11can be introduced into the human framework regions when such amino acidsare typical of human immunoglobulin at the equivalent positions.

Other candidates for substitution are non-germline residues occurring ina framework region. By performing a computer comparison of 12A11 withknown germline sequences, germline sequences with the greatest degree ofsequence identity to the heavy or light chain can be identified.Alignment of the framework region and the germline sequence will revealwhich residues may be selected for substitution with correspondinggermline residues. Residues not matching between a selected light chainacceptor framework and one of these germline sequences could be selectedfor substitution with the corresponding germline residue.

Rare mouse residues are identified by comparing the donor VL and/or VHsequences with the sequences of other members of the subgroup to whichthe donor VL and/or VH sequences belong (according to Kabat) andidentifying the residue positions which differ from the consensus. Thesedonor specific differences may point to somatic mutations which enhanceactivity. Unusual or rare residues close to the binding site maypossibly contact the antigen, making it desirable to retain the mouseresidue. However, if the unusual mouse residue is not important forbinding, use of the corresponding acceptor residue is preferred as themouse residue may create immunogenic neoepitopes in the humanizedantibody. In the situation where an unusual residue in the donorsequence is actually a common residue in the corresponding acceptorsequence, the preferred residue is clearly the acceptor residue.

Table 1A summarizes the sequence analysis of the 12A11 VH and VLregions. TABLE 1 Summary of 12A11 V region sequence Chain VL VH MouseSubgroup II Ib Human Subgroup II II Rare amino acids in mouse vk I85(3.6%) I11 (1.7%) (% frequency) Chothia canonical class L1: class 4[16f]H1: class 3 [7] L2: class 1[7] H2: class 1[16] L3: class 1[9] H3¹Closest mouse MAb solved 1KTR² 1ETZ³ (2.6 Å) and structure 1JRH⁴Homology with Modeling 94% 83% and 86% template Human Framework seq K64(BAC01733) M72 (AAA69734) (87% FR, 67% overall) (61% FR, 45% overall)Donornotes Hu k LC subgroup II HU HC subgroup III CDRs from same CDRsfrom same canonical canonical structural group Structural group as 12A11as 12A11 Backmutation Notes none A24F, F29L: H1 R71K: Canonical, H2V371: Packing T28S, V48L, F67L, N73T, L78V: Vernier Germline ref for HuFr A19 VL Vk2-28 AAA69731.1 mRNA: X63397.1 (GI: 567123) (GI: 33774)¹No canonical class but might form a kinked base according to the rulesof Shirai et al. (1999) FEBS Lett. 4 55: 188-197.²Kaufmann et al. (2002) J Mol Biol. 318: 135-147.³Guddat et al. (2000) J Mol Biol. 302: 853-872.⁴Sogabe et al. (1997) J Mol Biol. 273: 882-897.

Germline sequences are set forth that can be used in selecting aminoacid substitutions.

Three-dimensional structural information for antibodies described hereinis publicly available, for example, from the Research Collaboratory forStructural Bioinformatics' Protein Data Bank (PDB). The PDB is freelyaccessible via the World Wide Web internet and is described by Berman etal. (2000) Nucleic Acids Research, p235-242. Germline gene sequencesreferenced herein are publicly available, for example, from the NationalCenter for Biotechnology Information (NCBI) database of sequences incollections of Igh, Ig kappa and Ig lambda germline V genes (as adivision of the National Library of Medicine (NLM) at the NationalInstitutes of Health (NIH)). Homology searching of the NCBI “Ig GermlineGenes” database is provided by IgG BLAST™.

In an exemplary embodiment, a humanized antibody of the presentinvention contains (i) a light chain comprising a variable domaincomprising murine 12A11 VL CDRs and a human acceptor framework, theframework having zero, one, two, three, four, five, six, seven, eight,nine or more residues substituted with the corresponding 12A11 residueand (ii) a heavy chain comprising 12A11 VH CDRs and a human acceptorframework, the framework having at least one, two, three, four, five,six, seven, eight, nine or more residues substituted with thecorresponding 12A11 residue, and, optionally, at least one, preferablytwo or three residues substituted with a corresponding human germlineresidue.

In another exemplary embodiment, a humanized antibody of the presentinvention contains (i) a light chain comprising a variable domaincomprising murine 12A11 VL CDRs and a human acceptor framework, theframework having at least one, two, three, four, five, six, seven,eight, nine or more residues backmutated (i.e., substituted with thecorresponding 12A11 residue), wherein the backmutation(s) are at acanonical, packing and/or vernier residues and (ii) a heavy chaincomprising 12A11 VH CDRs and a human acceptor framework, the frameworkhaving at least one, two, three, four, five, six, seven, eight, nine ormore residues bacmutated, wherein the backmutation(s) are at acanonical, packing and/or vernier residues. In certain embodiments,backmutations are only at packing and/or canonical residues or areprimarily at canonical and/or packing residues (e.g., only 1 or 2vernier residues of the vernier residues differing between the donor andacceptor sequence are backmutated).

In other embodiments, humanized antibodies include the fewest number ofbackmutations possible while retaining a binding affinity comparable tothat of the donor antibody (or a chimeric version thereof). To arrive atsuch versions, various combinations of backmutations can be eliminatedand the resulting antibodies tested for efficacy (e.g., bindingaffinity). For example, backmutations (e.g., 1, 2, 3, or 4backmutations) at vernier residues can be eliminated or backmutations atcombinations of vernier and packing, vernier and canonical or packingand canonical residues can be eliminated.

In another embodiment, a humanized antibody of the present invention hasstructural features, as described herein, and further has at least one(preferably two, three, four or all) of the following activities: (1)binds soluble Aβ; (2) binds aggregated Aβ1-42 (e.g., as determined byELISA); (3) captures soluble Aβ; (4) binds Aβ in plaques (e.g., stainingof AD and/or PDAPP plaques); (5) binds Aβ with an affinity no less thantwo to three fold lower than chimeric 12A11 (e.g., 12A11 having murinevariable region sequences and human constant region sequences); (6)mediates phagocytosis of Aβ (e.g., in an ex vivo phagocytosis assay, asdescribed herein); and (7) crosses the blood-brain barrier (e.g.,demonstrates short-term brain localization, for example, in a PDAPPanimal model, as described herein).

In another embodiment, a humanized antibody of the present invention hasstructural features, as described herein, such that it binds Aβ in amanner or with an affinity sufficient to elicit at least one of thefollowing in vivo effects: (1) reduce Aβ plaque burden; (2) preventplaque formation; (3) reduce levels of soluble Aβ; (4) reduce theneuritic pathology associated with an amyloidogenic disorder; (5) lessenor ameliorate at least one physiological symptom associated with anamyloidogenic disorder; and/or (6) improve cognitive function.

In another embodiment, a humanized antibody of the present invention hasstructural features, as described herein, and specifically binds to anepitope comprising residues 3-7 of Aβ.

In yet another embodiment, a humanized antibody of the present inventionhas structural features, as described herein, such that it binds to anN-terminal epitope within Aβ (e.g., binds to an epitope within aminoacids 3-7 of Aβ), and is capable of reducing (1) Aβ peptide levels; (2)Aβ plaque burden; and (3) the neuritic burden or neuritic dystrophyassociated with an amyloidogenic disorder.

The activities described above can be determined utilizing any one of avariety of assays described herein or in the art (e.g., binding assays,phagocytosis assays, etc.). Activities can be assayed either in vivo(e.g., using labeled assay components and/or imaging techniques) or invitro (e.g., using samples or specimens derived from a subject).Activities can be assayed either directly or indirectly. In certainpreferred embodiments, neurological endpoints (e.g., amyloid burden,neuritic burden, etc) are assayed. Such endpoints can be assayed inliving subjects (e.g., in animal models of Alzheimer's disease or inhuman subjects, for example, undergoing immunotherapy) usingnon-invasive detection methodologies. Alternatively, such endpoints canbe assayed in subjects post mortem. Assaying such endpoints in animalmodels and/or in human subjects post mortem is useful in assessing theeffectiveness of various agents (e.g., humanized antibodies) to beutilized in similar immunotherapeutic applications. In other preferredembodiments, behavioral or neurological parameters can be assessed asindicators of the above neuropathological activities or endpoints.

3. Production of Variable Regions

Having conceptually selected the CDR and framework components ofhumanized immunoglobulins, a variety of methods are available forproducing such immunoglobulins. In general, one or more of the murinecomplementarity determining regions (CDR) of the heavy and/or lightchain of the antibody can be humanized, for example, placed in thecontext of one or more human framework regions, using primer-basedpolymerase chain reaction (PCR). Briefly, primers are designed which arecapable of annealing to target murine CDR region(s) which also containsequence which overlaps and can anneal with a human framework region.Accordingly, under appropriate conditions, the primers can amplify amurine CDR from a murine antibody template nucleic acid and add to theamplified template a portion of a human framework sequence. Similarly,primers can be designed which are capable of annealing to a target humanframework region(s) where a PCR reaction using these primers results inan amplified human framework region(s). When each amplification productis then denatured, combined, and annealed to the other product, themurine CDR region, having overlapping human framework sequence with theamplified human framework sequence, can be genetically linked.Accordingly, in one or more such reactions, one or more murine CDRregions can be genetically linked to intervening human frameworkregions.

In some embodiments, the primers may also comprise desirable restrictionenzyme recognition sequences to facilitate the genetic engineering ofthe resultant PCR amplified sequences into a larger genetic segment, forexample, a variable light or heavy chain segment, heavy chain, orvector. In addition, the primers used to amplify either the murine CDRregions or human framework regions may have desirable mismatches suchthat a different codon is introduced into the murine CDR or humanframework region. Typical mismatches introduce alterations in the humanframework regions that preserve or improve the structural orientation ofthe murine CDR and thus its binding affinity, as described herein.

It should be understood that the foregoing approach can be used tointroduce one, two, or all three murine CDR regions into the context ofintervening human framework regions. Methods for amplifying and linkingdifferent sequences using primer-based PCR are described in, forexample, Sambrook, Fritsch and Maniatis, Molecular Cloning: Cold SpringHarbor Laboratory Press (1989); DNA Cloning, Vols. 1 and 2, (D. N.Glover, Ed. 1985); PCR Handbook Current Protocols in Nucleic AcidChemistry, Beaucage, Ed. John Wiley & Sons (1999) (Editor); CurrentProtocols in Molecular Biology, eds. Ausubel et al., John Wiley & Sons(1992).

Because of the degeneracy of the code, a variety of nucleic acidsequences will encode each immunoglobulin amino acid sequence. Thedesired nucleic acid sequences can be produced by de novo solid-phaseDNA synthesis or by PCR mutagenesis of an earlier prepared variant ofthe desired polynucleotide. Oligonucleotide-mediated mutagenesis is apreferred method for preparing substitution, deletion and insertionvariants of target polypeptide DNA. See Adelman et al., DNA 2:183(1983). Briefly, the target polypeptide DNA is altered by hybridizing anoligonucleotide encoding the desired mutation to a single-stranded DNAtemplate. After hybridization, a DNA polymerase is used to synthesize anentire second complementary strand of the template that incorporates theoligonucleotide primer, and encodes the selected alteration in thetarget polypeptide DNA.

4. Selection of Constant Regions

The variable segments of antibodies produced as described supra (e.g.,the heavy and light chain variable regions of chimeric or humanizedantibodies) are typically linked to at least a portion of animmunoglobulin constant region (Fc region), typically that of a humanimmunoglobulin. Human constant region DNA sequences can be isolated inaccordance with well known procedures from a variety of human cells, butpreferably immortalized B cells (see Kabat et al., supra, and Liu etal., WO87/02671) (each of which is incorporated by reference in itsentirety for all purposes). Ordinarily, the antibody will contain bothlight chain and heavy chain constant regions. The heavy chain constantregion usually includes CH1, hinge, CH2, CH3, and CH4 regions. Theantibodies described herein include antibodies having all types ofconstant regions, including IgM, IgG, IgD, IgA and IgE, and any isotype,including IgG1, IgG2, IgG3 and IgG4. When it is desired that theantibody (e.g., humanized antibody) exhibit cytotoxic activity, theconstant domain is usually a complement fixing constant domain and theclass is typically IgG1. Human isotype IgG1 is preferred. Light chainconstant regions can be lambda or kappa. The humanized antibody maycomprise sequences from more than one class or isotype. Antibodies canbe expressed as tetramers containing two light and two heavy chains, asseparate heavy chains, light chains, as Fab, Fab′ F(ab′)2, and Fv, or assingle chain antibodies in which heavy and light chain variable domainsare linked through a spacer.

5. Expression of Recombinant Antibodies

Chimeric and humanized antibodies are typically produced by recombinantexpression. Nucleic acids encoding light and heavy chain variableregions, optionally linked to constant regions, are inserted intoexpression vectors. The light and heavy chains can be cloned in the sameor different expression vectors. The DNA segments encodingimmunoglobulin chains are operably linked to control sequences in theexpression vector(s) that ensure the expression of immunoglobulinpolypeptides. Expression control sequences include, but are not limitedto, promoters (e.g., naturally-associated or heterologous promoters),signal sequences, enhancer elements, and transcription terminationsequences. Preferably, the expression control sequences are eukaryoticpromoter systems in vectors capable of transforming or transfectingeukaryotic host cells (e.g., COS cells). Once the vector has beenincorporated into the appropriate host, the host is maintained underconditions suitable for high level expression of the nucleotidesequences, and the collection and purification of the crossreactingantibodies.

These expression vectors are typically replicable in the host organismseither as episomes or as an integral part of the host chromosomal DNA.Commonly, expression vectors contain selection markers (e.g.,ampicillin-resistance, hygromycin-resistance, tetracycline resistance,kanamycin resistance or neomycin resistance) to permit detection ofthose cells transformed with the desired DNA sequences (see, e.g.,Itakura et al., U.S. Pat. No. 4,704,362).

E. coli is one prokaryotic host particularly useful for cloning thepolynucleotides (e.g., DNA sequences) of the present invention. Othermicrobial hosts suitable for use include bacilli, such as Bacillussubtilis, and other enterobacteriaceae, such as Salmonella, Serratia,and various Pseudomonas species. In these prokaryotic hosts, one canalso make expression vectors, which will typically contain expressioncontrol sequences compatible with the host cell (e.g., an origin ofreplication). In addition, any number of a variety of well-knownpromoters will be present, such as the lactose promoter system, atryptophan (trp) promoter system, a beta-lactamase promoter system, or apromoter system from phage lambda. The promoters will typically controlexpression, optionally with an operator sequence, and have ribosomebinding site sequences and the like, for initiating and completingtranscription and translation.

Other microbes, such as yeast, are also useful for expression.Saccharomyces is a preferred yeast host, with suitable vectors havingexpression control sequences (e.g., promoters), an origin ofreplication, termination sequences and the like as desired. Typicalpromoters include 3-phosphoglycerate kinase and other glycolyticenzymes. Inducible yeast promoters include, among others, promoters fromalcohol dehydrogenase, isocytochrome C, and enzymes responsible formaltose and galactose utilization.

In addition to microorganisms, mammalian tissue cell culture may also beused to express and produce the polypeptides of the present invention(e.g., polynucleotides encoding immunoglobulins or fragments thereof).See Winnacker, From Genes to Clones, VCH Publishers, N.Y., N.Y. (1987).Eukaryotic cells are actually preferred, because a number of suitablehost cell lines capable of secreting heterologous proteins (e.g., intactimmunoglobulins) have been developed in the art, and include CHO celllines, various Cos cell lines, HeLa cells, preferably, myeloma celllines, or transformed B-cells or hybridomas. Preferably, the cells arenonhuman. Expression vectors for these cells can include expressioncontrol sequences, such as an origin of replication, a promoter, and anenhancer (Queen et al., Immunol. Rev. 89:49 (1986)), and necessaryprocessing information sites, such as ribosome binding sites, RNA splicesites, polyadenylation sites, and transcriptional terminator sequences.Preferred expression control sequences are promoters derived fromimmunoglobulin genes, SV40, adenovirus, bovine papilloma virus,cytomegalovirus and the like. See Co et al., J. Immunol. 148:1149(1992).

Alternatively, antibody-coding sequences can be incorporated intransgenes for introduction into the genome of a transgenic animal andsubsequent expression in the milk of the transgenic animal (see, e.g.,Deboer et al., U.S. Pat. No. 5,741,957, Rosen, U.S. Pat. No. 5,304,489,and Meade et al., U.S. Pat. No. 5,849,992). Suitable transgenes includecoding sequences for light and/or heavy chains in operable linkage witha promoter and enhancer from a mammary gland specific gene, such ascasein or beta lactoglobulin.

Alternatively, antibodies (e.g., humanized antibodies) of the inventioncan be produced in transgenic plants (e.g., tobacco, maize, soybean andalfalfa). Improved ‘plantibody’ vectors (Hendy et al. (1999) J. Immunol.Methods 231:137-146) and purification strategies coupled with anincrease in transformable crop species render such methods a practicaland efficient means of producing recombinant immunoglobulins not onlyfor human and animal therapy, but for industrial applications as well(e.g., catalytic antibodies). Moreover, plant produced antibodies havebeen shown to be safe and effective and avoid the use of animal-derivedmaterials and therefore the risk of contamination with a transmissiblespongiform encephalopathy (TSE) agent. Further, the differences inglycosylation patterns of plant and mammalian cell-produced antibodieshave little or no effect on antigen binding or specificity. In addition,no evidence of toxicity or HAMA has been observed in patients receivingtopical oral application of a plant-derived secretory dimeric IgAantibody (see Larrick et al. (1998) Res. Immunol. 149:603-608).

Various methods may be used to express recombinant antibodies intransgenic plants. For example, antibody heavy and light chains can beindependently cloned into expression vectors (e.g., Agrobacteriumtumefaciens vectors), followed by the transformation of plant tissue invitro with the recombinant bacterium or direct transformation using,e.g., particles coated with the vector which are then physicallyintroduced into the plant tissue using, e.g., ballistics. Subsequently,whole plants expressing individual chains are reconstituted followed bytheir sexual cross, ultimately resulting in the production of a fullyassembled and functional antibody. Similar protocols have been used toexpress functional antibodies in tobacco plants (see Hiatt et al. (1989)Nature 342:76-87). In various embodiments, signal sequences may beutilized to promote the expression, binding and folding of unassembledantibody chains by directing the chains to the appropriate plantenvironment (e.g., the aqueous environment of the apoplasm or otherspecific plant tissues including tubers, fruit or seed) (see Fiedler etal. (1995) Bio/Technology 13:1090-1093). Plant bioreactors can also beused to increase antibody yield and to significantly reduce costs.

The vectors containing the polynucleotide sequences of interest (e.g.,the heavy and light chain encoding sequences and expression controlsequences) can be transferred into the host cell by well-known methods,which vary depending on the type of cellular host. For example, calciumchloride transfection is commonly utilized for prokaryotic cells,whereas calcium phosphate treatment, electroporation, lipofection,biolistics or viral-based transfection may be used for other cellularhosts. (See generally Sambrook et al., Molecular Cloning: A LaboratoryManual (Cold Spring Harbor Press, 2nd ed., 1989) (incorporated byreference in its entirety for all purposes). Other methods used totransform mammalian cells include the use of polybrene, protoplastfusion, liposomes, electroporation, and microinjection (see generally,Sambrook et al., supra). For production of transgenic animals,transgenes can be microinjected into fertilized oocytes, or can beincorporated into the genome of embryonic stem cells, and the nuclei ofsuch cells transferred into enucleated oocytes.

When heavy and light chains are cloned on separate expression vectors,the vectors are co-transfected to obtain expression and assembly ofintact immunoglobulins. Once expressed, the whole antibodies, theirdimers, individual light and heavy chains, or other immunoglobulin formsof the present invention can be purified according to standardprocedures of the art, including ammonium sulfate precipitation,affinity columns, column chromatography, HPLC purification, gelelectrophoresis and the like (see generally Scopes, Protein Purification(Springer-Verlag, N.Y., (1982)). Substantially pure immunoglobulins ofat least about 90 to 95% homogeneity are preferred, and 98 to 99% ormore homogeneity most preferred, for pharmaceutical uses.

6. Antibody Fragments

Also contemplated within the scope of the instant invention are antibodyfragments. In one embodiment, fragments of non-human, and/or chimericantibodies are provided. In another embodiment, fragments of humanizedantibodies are provided. Typically, these fragments exhibit specificbinding to antigen with an affinity of at least 10⁷, and more typically10⁸ or 10⁹ M⁻¹. Humanized antibody fragments include separate heavychains, light chains, Fab, Fab′, F(ab′)2, Fabc, and Fv. Fragments areproduced by recombinant DNA techniques, or by enzymatic or chemicalseparation of intact immunoglobulins.

7. Epitope Mapping

Epitope mapping can be performed to determine which antigenicdeterminant or epitope of Aβ is recognized by the antibody. In oneembodiment, epitope mapping is performed according to Replacement NET(rNET) analysis. The rNET epitope map assay provides information aboutthe contribution of individual residues within the epitope to theoverall binding activity of the antibody. rNET analysis uses synthesizedsystematic single substituted peptide analogs. Binding of an antibodybeing tested is determined against native peptide (native antigen) andagainst 19 alternative “single substituted” peptides, each peptide beingsubstituted at a first position with one of 19 non-native amino acidsfor that position. A profile is generated reflecting the effect ofsubstitution at that position with the various non-native residues.Profiles are likewise generated at successive positions along theantigenic peptide. The combined profile, or epitope map, (reflectingsubstitution at each position with all 19 non-native residues) can thenbe compared to a map similarly generated for a second antibody.Substantially similar or identical maps indicate that antibodies beingcompared have the same or similar epitope specificity.

8. Testing Antibodies for Therapeutic Efficacy in Animal Models

Groups of 7-9 month old PDAPP mice each are injected with 0.5 mg in PBSof polyclonal anti-Aβ or specific anti-Aβ monoclonal antibodies. Allantibody preparations are purified to have low endotoxin levels.Monoclonals can be prepared against a fragment by injecting the fragmentor longer form of Aβ into a mouse, preparing hybridomas and screeningthe hybridomas for an antibody that specifically binds to a desiredfragment of Aβ without binding to other nonoverlapping fragments of Aβ.

Mice are injected intraperitoneally as needed over a 4 month period tomaintain a circulating antibody concentration measured by ELISA titer ofgreater than {fraction (1/1000)} defined by ELISA to Aβ42 or otherimmunogen. Titers are monitored and mice are euthanized at the end of 6months of injections. Histochemistry, Aβ levels and toxicology areperformed post mortem. Ten mice are used per group.

9. Screening Antibodies for Clearing Activity

The invention also provides methods of screening an antibody foractivity in clearing an amyloid deposit or any other antigen, orassociated biological entity, for which clearing activity is desired. Toscreen for activity against an amyloid deposit, a tissue sample from abrain of a patient with Alzheimer's disease or an animal model havingcharacteristic Alzheimer's pathology is contacted with phagocytic cellsbearing an Fc receptor, such as microglial cells, and the antibody undertest in a medium in vitro. The phagocytic cells can be a primary cultureor a cell line, and can be of murine (e.g., BV-2 or C8-B4 cells) orhuman origin (e.g., THP-1 cells). In some methods, the components arecombined on a microscope slide to facilitate microscopic monitoring. Insome methods, multiple reactions are performed in parallel in the wellsof a microtiter dish. In such a format, a separate miniature microscopeslide can be mounted in the separate wells, or a nonmicroscopicdetection format, such as ELISA detection of Aβ can be used. Preferably,a series of measurements is made of the amount of amyloid deposit in thein vitro reaction mixture, starting from a baseline value before thereaction has proceeded, and one or more test values during the reaction.The antigen can be detected by staining, for example, with afluorescently labeled antibody to Aβ or other component of amyloidplaques. The antibody used for staining may or may not be the same asthe antibody being tested for clearing activity. A reduction relative tobaseline during the reaction of the amyloid deposits indicates that theantibody under test has clearing activity. Such antibodies are likely tobe useful in preventing or treating Alzheimer's and other amyloidogenicdiseases. Particularly useful antibodies for preventing or treatingAlzheimer's and other amyloidogenic diseases include those capable ofclearing both compact and diffuse amyloid plaques, for example, the12A11 antibody of the instant invention, or chimeric or humanizedversions thereof.

Analogous methods can be used to screen antibodies for activity inclearing other types of biological entities. The assay can be used todetect clearing activity against virtually any kind of biologicalentity. Typically, the biological entity has some role in human oranimal disease. The biological entity can be provided as a tissue sampleor in isolated form. If provided as a tissue sample, the tissue sampleis preferably unfixed to allow ready access to components of the tissuesample and to avoid perturbing the conformation of the componentsincidental to fixing. Examples of tissue samples that can be tested inthis assay include cancerous tissue, precancerous tissue, tissuecontaining benign growths such as warts or moles, tissue infected withpathogenic microorganisms, tissue infiltrated with inflammatory cells,tissue bearing pathological matrices between cells (e.g., fibrinouspericarditis), tissue bearing aberrant antigens, and scar tissue.Examples of isolated biological entities that can be used include Aβ,viral antigens or viruses, proteoglycans, antigens of other pathogenicmicroorganisms, tumor antigens, and adhesion molecules. Such antigenscan be obtained from natural sources, recombinant expression or chemicalsynthesis, among other means. The tissue sample or isolated biologicalentity is contacted with phagocytic cells bearing Fc receptors, such asmonocytes or microglial cells, and an antibody to be tested in a medium.The antibody can be directed to the biological entity under test or toan antigen associated with the entity. In the latter situation, theobject is to test whether the biological entity is phagocytosed with theantigen. Usually, although not necessarily, the antibody and biologicalentity (sometimes with an associated antigen), are contacted with eachother before adding the phagocytic cells. The concentration of thebiological entity and/or the associated antigen remaining in the medium,if present, is then monitored. A reduction in the amount orconcentration of antigen or the associated biological entity in themedium indicates the antibody has a clearing response against theantigen and/or associated biological entity in conjunction with thephagocytic cells.

10. Chimeric/Humanized Antibodies Having Altered Effector Function

For the above-described antibodies of the invention comprising aconstant region (Fc region), it may also be desirable to alter theeffector function of the molecule. Generally, the effector function ofan antibody resides in the constant or Fc region of the molecule whichcan mediate binding to various effector molecules, e.g., complementproteins or Fc receptors. The binding of complement to the Fc region isimportant, for example, in the opsonization and lysis of cell pathogensand the activation of inflammatory responses. The binding of antibody toFc receptors, for example, on the surface of effector cells can triggera number of important and diverse biological responses including, forexample, engulfment and destruction of antibody-coated pathogens orparticles, clearance of immune complexes, lysis of antibody-coatedtarget cells by killer cells (i.e., antibody-dependent cell-mediatedcytotoxicity, or ADCC), release of inflammatory mediators, placentaltransfer of antibodies, and control of immunoglobulin production.

Accordingly, depending on a particular therapeutic or diagnosticapplication, the above-mentioned immune functions, or only selectedimmune functions, may be desirable. By altering the Fc region of theantibody, various aspects of the effector function of the molecule,including enhancing or suppressing various reactions of the immunesystem, with beneficial effects in diagnosis and therapy, are achieved.

The antibodies of the invention can be produced which react only withcertain types of Fc receptors, for example, the antibodies of theinvention can be modified to bind to only certain Fc receptors, or ifdesired, lack Fc receptor binding entirely, by deletion or alteration ofthe Fc receptor binding site located in the Fc region of the antibody.Other desirable alterations of the Fc region of an antibody of theinvention are cataloged below. Typically the Kabat numbering system isused to indicate which amino acid residue(s) of the Fc region (e.g., ofan IgG antibody) are altered (e.g., by amino acid substitution) in orderto achieve a desired change in effector function. The numbering systemis also employed to compare antibodies across species such that adesired effector function observed in, for example, a mouse antibody,can then be systematically engineered into a human, humanized, orchimeric antibody of the invention.

For example, it has been observed that antibodies (e.g., IgG antibodies)can be grouped into those found to exhibit tight, intermediate, or weakbinding to an Fc receptor (e.g., an Fc receptor on human monocytes(FcγRI)). By comparison of the amino-acid sequences in these differentaffinity groups, a monocyte-binding site in the hinge-link region(Leu234-Ser239) has been identified. Moreover, the human FcγRI receptorbinds human IgG1 and mouse IgG2a as a monomer, but the binding of mouseIgG2b is 100-fold weaker. A comparison of the sequence of these proteinsin the hinge-link region shows that the sequence 234 to 238, i.e.,Leu-Leu-Gly-Gly-Pro (SEQ ID NO:32) in the strong binders becomesLeu-Glu-Gly-Gly-Pro (SEQ ID NO:33) in mouse gamma 2b, i.e., weakbinders. Accordingly, a corresponding change in a human antibody hingesequence can be made if reduced FcγI receptor binding is desired. It isunderstood that other alterations can be made to achieve the same orsimilar results. For example, the affinity of FcγRI binding can bealtered by replacing the specified residue with a residue having aninappropriate functional group on its sidechain, or by introducing acharged functional group (e.g., Glu or Asp) or for example an aromaticnon-polar residue (e.g., Phe, Tyr, or Trp).

These changes can be equally applied to the murine, human, and ratsystems given the sequence homology between the differentimmunoglobulins. It has been shown that for human IgG3, which binds tothe human FcγRI receptor, changing Leu 235 to Glu destroys theinteraction of the mutant for the receptor. The binding site for thisreceptor can thus be switched on or switched off by making theappropriate mutation.

Mutations on adjacent or close sites in the hinge link region (e.g.,replacing residues 234, 236 or 237 by Ala) indicate that alterations inresidues 234, 235, 236, and 237 at least affect affinity for the FcγRIreceptor. Accordingly, the antibodies of the invention can also have analtered Fc region with altered binding affinity for FcγRI as comparedwith the unmodified antibody. Such an antibody conveniently has amodification at amino acid residue 234, 235, 236, or 237.

Affinity for other Fc receptors can be altered by a similar approach,for controlling the immune response in different ways.

As a further example, the lytic properties of IgG antibodies followingbinding of the Cl component of complement can be altered.

The first component of the complement system, Cl, comprises threeproteins known as Clq, Clr and Cls which bind tightly together. It hasbeen shown that Clq is responsible for binding of the three proteincomplex to an antibody.

Accordingly, the Clq binding activity of an antibody can be altered byproviding an antibody with an altered CH 2 domain in which at least oneof the amino acid residues 318, 320, and 322 of the heavy chain has beenchanged to a residue having a different side chain. The numbering of theresidues in the heavy chain is that of the EU index (see Kabat et al.,supra). Other suitable alterations for altering, e.g., reducing orabolishing specific Clq-binding to an antibody include changing any oneof residues 318 (Glu), 320 (Lys) and 322 (Lys), to Ala.

Moreover, by making mutations at these residues, it has been shown thatClq binding is retained as long as residue 318 has a hydrogen-bondingside chain and residues 320 and 322 both have a positively charged sidechain.

Clq binding activity can be abolished by replacing any one of the threespecified residues with a residue having an inappropriate functionalityon its side chain. It is not necessary to replace the ionic residuesonly with Ala to abolish Clq binding. It is also possible to use otheralkyl-substituted non-ionic residues, such as Gly, Ile, Leu, or Val, orsuch aromatic non-polar residues as Phe, Tyr, Trp and Pro in place ofany one of the three residues in order to abolish Clq binding. Inaddition, it is also be possible to use such polar non-ionic residues asSer, Thr, Cys, and Met in place of residues 320 and 322, but not 318, inorder to abolish Clq binding activity.

It is also noted that the side chains on ionic or non-ionic polarresidues will be able to form hydrogen bonds in a similar manner to thebonds formed by the Glu residue. Therefore, replacement of the 318 (Glu)residue by a polar residue may modify but not abolish Clq bindingactivity.

It is also known that replacing residue 297 (Asn) with Ala results inremoval of lytic activity while only slightly reducing (about three foldweaker) affinity for Clq. This alteration destroys the glycosylationsite and the presence of carbohydrate that is required for complementactivation. Any other substitution at this site will also destroy theglycosylation site.

The invention also provides an antibody having an altered effectorfunction wherein the antibody has a modified hinge region. The modifiedhinge region may comprise a complete hinge region derived from anantibody of different antibody class or subclass from that of the CH1domain. For example, the constant domain (CH1) of a class IgG antibodycan be attached to a hinge region of a class IgG4 antibody.Alternatively, the new hinge region may comprise part of a natural hingeor a repeating unit in which each unit in the repeat is derived from anatural hinge region. In one example, the natural hinge region isaltered by converting one or more cysteine residues into a neutralresidue, such as alanine, or by converting suitably placed residues intocysteine residues. Such alterations are carried out using art recognizedprotein chemistry and, preferably, genetic engineering techniques, asdescribed herein.

In one embodiment of the invention, the number of cysteine residues inthe hinge region of the antibody is reduced, for example, to onecysteine residue. This modification has the advantage of facilitatingthe assembly of the antibody, for example, bispecific antibody moleculesand antibody molecules wherein the Fc portion has been replaced by aneffector or reporter molecule, since it is only necessary to form asingle disulfide bond. This modification also provides a specific targetfor attaching the hinge region either to another hinge region or to aneffector or reporter molecule, either directly or indirectly, forexample, by chemical means.

Conversely, the number of cysteine residues in the hinge region of theantibody is increased, for example, at least one more than the number ofnormally occurring cysteine residues. Increasing the number of cysteineresidues can be used to stabilize the interactions between adjacenthinges. Another advantage of this modification is that it facilitatesthe use of cysteine thiol groups for attaching effector or reportermolecules to the altered antibody, for example, a radiolabel.

Accordingly, the invention provides for an exchange of hinge regionsbetween antibody classes, in particular, IgG classes, and/or an increaseor decrease in the number of cysteine residues in the hinge region inorder to achieve an altered effector function (see for example U.S. Pat.No. 5,677,425 which is expressly incorporated herein). A determinationof altered antibody effector function is made using the assays describedherein or other art recognized techniques.

Importantly, the resultant antibody can be subjected to one or moreassays to evaluate any change in biological activity compared to thestarting antibody. For example, the ability of the antibody with analtered Fc region to bind complement or Fc receptors can be assessedusing the assays disclosed herein as well as any art recognized assay.

Production of the antibodies of the invention is carried out by anysuitable technique including techniques described herein as well astechniques known to those skilled in the art. For example an appropriateprotein sequence, e.g. forming part of or all of a relevant constantdomain, e.g., Fc region, i.e., CH2, and/or CH3 domain(s), of anantibody, and include appropriately altered residue(s) can besynthesized and then chemically joined into the appropriate place in anantibody molecule.

Preferably, genetic engineering techniques are used for producing analtered antibody. Preferred techniques include, for example, preparingsuitable primers for use in polymerase chain reaction (PCR) such that aDNA sequence which encodes at least part of an IgG heavy chain, e.g., anFc or constant region (e.g., CH2, and/or CH3) is altered, at one or moreresidues. The segment can then be operably linked to the remainingportion of the antibody, e.g., the variable region of the antibody andrequired regulatory elements for expression in a cell.

The present invention also includes vectors used to transform the cellline, vectors used in producing the transforming vectors, cell linestransformed with the transforming vectors, cell lines transformed withpreparative vectors, and methods for their production.

Preferably, the cell line which is transformed to produce the antibodywith an altered Fc region (i.e., of altered effector function) is animmortalized mammalian cell line (e.g., CHO cell).

Although the cell line used to produce the antibody with an altered Fcregion is preferably a mammalian cell line, any other suitable cellline, such as a bacterial cell line or a yeast cell line, mayalternatively be used.

11. Affinity Maturation

Antibodies (e.g., humanized antibodies) of the invention can be modifiedfor improved function using any of a number of affinity maturationtechniques. Typically, a candidate molecule with a binding affinity to agiven target molecule is identified and then further improved or“matured” using mutagenesis techniques resulting in one or more relatedcandidates having a more desired binding interaction with the targetmolecule. Typically, it is the affinity of the antibody (or avidity,i.e., the combined affinities of the antibody for a target antigen) thatis modified, however, other properties of the molecule, such asstability, effector function, clearance, secretion, or transportfunction, may also be modified, either separately or in parallel withaffinity, using affinity maturation techniques.

In exemplary embodiments, the affinity of an antibody (e.g., a humanizedantibody of the instant invention) is increased. For example, antibodieshaving binding affinities of at least 10⁷M⁻¹, 10⁸M⁻¹ or 10⁹M⁻¹ can bematured such that their affinities are at least 10⁹M⁻¹, 10¹⁰M⁻¹ or 10¹²M⁻¹.

One approach for affinity maturing a binding molecule is to synthesize anucleic acid encoding the binding molecule, or portion thereof, thatencodes the desired change or changes. Oligonucleotide synthesis is wellknown in the art and readily automated to produce one or more nucleicacids having any desired codon change(s). Restriction sites, silentmutations, and favorable codon usage may also be introduced in this way.Alternatively, one or more codons can be altered to represent a subsetof particular amino acids, e.g., a subset that excludes cysteines whichcan form disulfide linkages, and is limited to a defined region, forexample, a CDR region or portion thereof. Alternatively, the region maybe represented by a partially or entirely random set of amino acids (foradditional details, see, e.g., U.S. Pat. Nos. 5,830,650; 5,798,208;5,824,514; 5,817,483; 5,814,476; 5,723,323; 4,528,266; 4,359,53;5,840,479; and 5,869,644).

It is understood that the above approaches can be carried out in part orin full using polymerase chain reaction (PCR) which is well known in theart and has the advantage of incorporating oligonucleotides, e.g.,primers or single stranded nucleic acids having, e.g., a desiredalteration(s), into a double stranded nucleic acid and in amplifiedamounts suitable for other manipulations, such as genetic engineeringinto an appropriate expression or cloning vector. Such PCR can also becarried out under conditions that allow for misincorporation ofnucleotides to thereby introduce additional variability into the nucleicacids being amplified. Experimental details for carrying out PCR andrelated kits, reagents, and primer design can be found, e.g., in U.S.Pat. Nos. 4,683,202; 4,683,195; 6,040,166; and 6,096,551. Methods forintroducing CDR regions into antibody framework regions usingprimer-based PCR is described in, e.g., U.S. Pat. No. 5,858,725. Methodsfor primer-based PCR amplification of antibody libraries (and librariesmade according to method) employing a minimal set of primers capable offinding sequence homology with a larger set of antibody molecules, suchthat a larger and diverse set of antibody molecules can be efficientlyamplified, is described, e.g., in U.S. Pat. Nos. 5,780,225; 6,303,313;and 6,479,243. Non PCR-based methods for performing site directedmutagenesis can also be used and include “Kunkel” mutagenesis thatemploys single-stranded uracil containing templates and primers thathybridize and introduce a mutation when passed through a particularstrain of E. coli (see, e.g., U.S. Pat. No. 4,873,192).

Additional methods for varying an antibody sequence, or portion thereof,include nucleic acid synthesis or PCR of nucleic acids under nonoptimal(i.e., error-prone) conditions, denaturation and renaturation(annealing) of such nucleic acids, exonuclease and/or endonucleasedigestion followed by reassembly by ligation or PCR (nucleic acidshuffling), or a combination of one or more of the foregoing techniquesas described, for example, in U.S. Pat. Nos. 6,440,668; 6,238,884;6,171,820; 5,965,408; 6,361,974; 6,358,709; 6,352,842; 4,888,286;6,337,186; 6,165,793; 6,132,970; 6,117,679; 5,830,721; and 5,605,793.

In certain embodiment, antibody libraries (or affinity maturationlibraries) comprising a family of candidate antibody molecules havingdiversity in certain portions of the candidate antibody molecule, e.g.,in one or more CDR regions (or a portion thereof), one or more frameworkregions, and/or one or more constant regions (e.g., a constant regionhaving effector function) can be expressed and screened for desiredproperties using art recognized techniques (see, e.g., U.S. Pat. Nos.6,291,161; 6,291,160; 6,291,159; and 6,291,158). For example, expressionlibraries of antibody variable domains having a diversity of CDR3sequences and methods for producing human antibody libraries having adiversity of CDR3 sequences by introducing, by mutagenesis, a diversityof CDR3 sequences and recovering the library can be constructed (see,e.g., U.S. Pat. No. 6,248,516).

Finally, for expressing the affinity matured antibodies, nucleic acidsencoding the candidate antibody molecules can be introduced into cellsin an appropriate expression format, e.g., as full length antibody heavyand light chains (e.g., IgG), antibody Fab fragments (e.g., Fab,F(ab′)₂), or as single chain antibodies (scFv) using standard vector andcell transfection/transformation technologies (see, e.g., U.S. Pat. Nos.6,331,415; 6,103,889; 5,260,203; 5,258,498; and 4,946,778).

B. Nucleic Acid Encoding Immunologic and Therapeutic Agents

Immune responses against amyloid deposits can also be induced byadministration of nucleic acids encoding antibodies and their componentchains used for passive immunization. Such nucleic acids can be DNA orRNA. A nucleic acid segment encoding an immunogen is typically linked toregulatory elements, such as a promoter and enhancer, that allowexpression of the DNA segment in the intended target cells of a patient.For expression in blood cells, as is desirable for induction of animmune response, exemplary promoter and enhancer elements include thosefrom light or heavy chain immunoglobulin genes and/or the CMV majorintermediate early promoter and enhancer (Stinski, U.S. Pat. Nos.5,168,062 and 5,385,839). The linked regulatory elements and codingsequences are often cloned into a vector. For administration ofdouble-chain antibodies, the two chains can be cloned in the same orseparate vectors.

A number of viral vector systems are available including retroviralsystems (see, e.g., Lawrie and Tumin, Cur. Opin. Genet. Develop.3:102-109 (1993)); adenoviral vectors (see, e.g., Bett et al., J. Virol.67:5911 (1993)); adeno-associated virus vectors (see, e.g., Zhou et al.,J. Exp. Med. 179:1867 (1994)), viral vectors from the pox familyincluding vaccinia virus and the avian pox viruses, viral vectors fromthe alpha virus genus such as those derived from Sindbis and SemlikiForest Viruses (see, e.g., Dubensky et al., J. Virol. 70:508 (1996)),Venezuelan equine encephalitis virus (see Johnston et al., U.S. Pat. No.5,643,576) and rhabdoviruses, such as vesicular stomatitis virus (seeRose, 6,168,943) and papillomaviruses (Ohe et al., Human Gene Therapy6:325 (1995); Woo et al., WO 94/12629 and Xiao & Brandsma, NucleicAcids. Res. 24, 2630-2622 (1996)).

DNA encoding an immunogen, or a vector containing the same, can bepackaged into liposomes. Suitable lipids and related analogs aredescribed by Eppstein et al., U.S. Pat. No. 5,208,036, Felgner et al.,U.S. Pat. No. 5,264,618, Rose, U.S. Pat. No. 5,279,833, and Epand etal., U.S. Pat. No. 5,283,185. Vectors and DNA encoding an immunogen canalso be adsorbed to or associated with particulate carriers, examples ofwhich include polymethyl methacrylate polymers and polylactides and poly(lactide-co-glycolides), see, e.g., McGee et al., J. Micro Encap.(1996).

Gene therapy vectors or naked polypeptides (e.g., DNA) can be deliveredin vivo by administration to an individual patient, typically bysystemic administration (e.g., intravenous, intraperitoneal, nasal,gastric, intradermal, intramuscular, subdermal, or intracranialinfusion) or topical application (see e.g., Anderson et al., U.S. Pat.No. 5,399,346). The term “naked polynucleotide” refers to apolynucleotide not delivered in association with a transfectionfacilitating agent. Naked polynucleotides are sometimes cloned in aplasmid vector. Such vectors can further include facilitating agentssuch as bupivacaine (Weiner et al., U.S. Pat. No. 5,593,972). DNA canalso be administered using a gene gun. See Xiao & Brandsma, supra. TheDNA encoding an immunogen is precipitated onto the surface ofmicroscopic metal beads. The microprojectiles are accelerated with ashock wave or expanding helium gas, and penetrate tissues to a depth ofseveral cell layers. For example, The Accel™ Gene Delivery Devicemanufactured by Agricetus, Inc. Middleton Wis. is suitable.Alternatively, naked DNA can pass through skin into the blood streamsimply by spotting the DNA onto skin with chemical or mechanicalirritation (see Howell et al., WO 95/05853).

In a further variation, vectors encoding immunogens can be delivered tocells ex vivo, such as-cells explanted from an individual patient (e.g.,lymphocytes, bone marrow aspirates, tissue biopsy) or universal donorhematopoietic stem cells, followed by reimplantation of the cells into apatient, usually after selection for cells which have incorporated thevector.

II. Prophylactic and Therapeutic Methods

The present invention is directed inter alia to treatment of Alzheimer'sand other amyloidogenic diseases by administration of therapeuticimmunological reagents (e.g., humanized immunoglobulins) to specificepitopes within Aβ to a patient under conditions that generate abeneficial therapeutic response in a patient (e.g., induction ofphagocytosis of Aβ, reduction of plaque burden, inhibition of plaqueformation, reduction of neuritic dystrophy, improving cognitivefunction, and/or reversing, treating or preventing cognitive decline) inthe patient, for example, for the prevention or treatment of anamyloidogenic disease. The invention is also directed to use of thedisclosed immunological reagents (e.g., humanized immunoglobulins) inthe manufacture of a medicament for the treatment or prevention of anamyloidogenic disease.

In one aspect, the invention provides methods of preventing or treatinga disease associated with amyloid deposits of Aβ in the brain of apatient. Such diseases include Alzheimer's disease, Down's syndrome andcognitive impairment. The latter can occur with or without othercharacteristics of an amyloidogenic disease. Some methods of theinvention comprise administering an effective dosage of an antibody thatspecifically binds to a component of an amyloid deposit to the patient.Such methods are particularly useful for preventing or treatingAlzheimer's disease in human patients. Exemplary methods compriseadministering an effective dosage of an antibody that binds to Aβ.Preferred methods comprise administering an effective dosage of anantibody that specifically binds to an epitope within residues 1-10 ofAβ, for example, antibodies that specifically bind to an epitope withinresidues 1-3 of Aβ, antibodies that specifically bind to an epitopewithin residues 1-4 of Aβ, antibodies that specifically bind to anepitope within residues 1-5 of Aβ, antibodies that specifically bind toan epitope within residues 1-6 of Aβ, antibodies that specifically bindto an epitope within residues 1-7 of Aβ, or antibodies that specificallybind to an epitope within residues 3-7 of Aβ. In yet another aspect, theinvention features administering antibodies that bind to an epitopecomprising a free N-terminal residue of Aβ. In yet another aspect, theinvention features administering antibodies that bind to an epitopewithin residues of 1-10 of Aβ wherein residue 1 and/or residue 7 of Aβis aspartic acid. In yet another aspect, the invention featuresadministering antibodies that specifically bind to Aβ peptide withoutbinding to full-length amyloid precursor protein (APP). In yet anotheraspect, the isotype of the antibody is human IgG1.

In yet another aspect, the invention features administering antibodiesthat bind to an amyloid deposit in the patient and induce a clearingresponse against the amyloid deposit. For example, such a clearingresponse can be effected by Fc receptor mediated phagocytosis.

Therapeutic agents of the invention are typically substantially purefrom undesired contaminant. This means that an agent is typically atleast about 50% w/w (weight/weight) pure, as well as being substantiallyfree from interfering proteins and contaminants. Sometimes the agentsare at least about 80% w/w and, more preferably at least 90 or about 95%w/w pure. However, using conventional protein purification techniques,homogeneous peptides of at least 99% w/w pure can be obtained.

The methods can be used on both asymptomatic patients and thosecurrently showing symptoms of disease. The antibodies used in suchmethods can be human, humanized, chimeric or nonhuman antibodies, orfragments thereof (e.g., antigen binding fragments) and can bemonoclonal or polyclonal, as described herein. In yet another aspect,the invention features administering antibodies prepared from a humanimmunized with Aβ peptide, which human can be the patient to be treatedwith antibody.

In another aspect, the invention features administering an antibody witha pharmaceutical carrier as a pharmaceutical composition. Alternatively,the antibody can be administered to a patient by administering apolynucleotide encoding at least one antibody chain. The polynucleotideis expressed to produce the antibody chain in the patient. Optionally,the polynucleotide encodes heavy and light chains of the antibody. Thepolynucleotide is expressed to produce the heavy and light chains in thepatient. In exemplary embodiments, the patient is monitored for level ofadministered antibody in the blood of the patient.

The invention thus fulfills a longstanding need for therapeutic regimesfor preventing or ameliorating the neuropathology and, in some patients,the cognitive impairment associated with Alzheimer's disease.

A. Patients Amenable to Treatment

Patients amenable to treatment include individuals at risk of diseasebut not showing symptoms, as well as patients presently showingsymptoms. In the case of Alzheimer's disease, virtually anyone is atrisk of suffering from Alzheimer's disease if he or she lives longenough. Therefore, the present methods can be administeredprophylactically to the general population without the need for anyassessment of the risk of the subject patient. The present methods areespecially useful for individuals who have a known genetic risk ofAlzheimer's disease. Such individuals include those having relatives whohave experienced this disease, and those whose risk is determined byanalysis of genetic or biochemical markers. Genetic markers of risktoward Alzheimer's disease include mutations in the APP gene,particularly mutations at position 717 and positions 670 and 671referred to as the Hardy and Swedish mutations respectively (see Hardy,supra). Other markers of risk are mutations in the presenilin genes, PS1and PS2, and ApoE4, family history of AD, hypercholesterolemia oratherosclerosis. Individuals presently suffering from Alzheimer'sdisease can be recognized from characteristic dementia, as well as thepresence of risk factors described above. In addition, a number ofdiagnostic tests are available for identifying individuals who have AD.These include measurement of CSF tau and Aβ42 levels. Elevated tau anddecreased Aβ42 levels signify the presence of AD. Individuals sufferingfrom Alzheimer's disease can also be diagnosed by ADRDA criteria asdiscussed in the Examples section.

In asymptomatic patients, treatment can begin at any age (e.g., 10, 20,30). Usually, however, it is not necessary to begin treatment until apatient reaches 40, 50, 60 or 70. Treatment typically involves multipledosages over a period of time. Treatment can be monitored by assayingantibody levels over time. If the response falls, a booster dosage isindicated. In the case of potential Down's syndrome patients, treatmentcan begin antenatally by administering therapeutic agent to the motheror shortly after birth.

B. Treatment Regimes and Dosages

In prophylactic applications, pharmaceutical compositions or medicamentsare administered to a patient susceptible to, or otherwise at risk of,Alzheimer's disease in an amount sufficient to eliminate or reduce therisk, lessen the severity, or delay the outset of the disease, includingbiochemical, histologic and/or behavioral symptoms of the disease, itscomplications and intermediate pathological phenotypes presenting duringdevelopment of the disease. In therapeutic applications, compositions ormedicaments are administered to a patient suspected of, or alreadysuffering from such a disease in an amount sufficient to cure, or atleast partially arrest, the symptoms of the disease (biochemical,histologic and/or behavioral), including its complications andintermediate pathological phenotypes in development of the disease.

In some methods, administration of agent reduces or eliminatesmyocognitive impairment in patients that have not yet developedcharacteristic Alzheimer's pathology. An amount adequate to accomplishtherapeutic or prophylactic treatment is defined as a therapeutically-or prophylactically-effective dose. In both prophylactic and therapeuticregimes, agents are usually administered in several dosages until asufficient immune response has been achieved. The term “immune response”or “immunological response” includes the development of a humoral(antibody mediated) and/or a cellular (mediated by antigen-specific Tcells or their secretion products) response directed against an antigenin a recipient subject. Such a response can be an active response, i.e.,induced by administration of immunogen, or a passive response, i.e.,induced by administration of immunoglobulin or antibody or primedT-cells. Typically, the immune response is monitored and repeated,dosages are given if the immune response starts to wane.

Effective doses of the compositions of the present invention, for thetreatment of the above described conditions vary depending upon manydifferent factors, including means of administration, target site,physiological state of the patient, whether the patient is human or ananimal, other medications administered, and whether treatment isprophylactic or therapeutic. Usually, the patient is a human butnon-human mammals including transgenic mammals can also be treated.Treatment dosages need to be titrated to optimize safety and efficacy.

For passive immunization with an antibody, the dosage ranges from about0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg (e.g., 0.02 mg/kg,0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 2 mg/kg, etc.), of the hostbody weight. For example dosages can be 1 mg/kg body weight or 10 mg/kgbody weight or within the range of 1-10 mg/kg, preferably at least 1mg/kg. In another example, dosages can be 0.5 mg/kg body weight or 15mg/kg body weight or within the range of 0.5-15 mg/kg, preferably atleast 1 mg/kg. Doses intermediate in the above ranges are also intendedto be within the scope of the invention. Subjects can be administeredsuch doses daily, on alternative days, weekly or according to any otherschedule determined by empirical analysis. An exemplary treatmentinvolves administration in multiple dosages over a prolonged period, forexample, of at least six months. Additional exemplary treatment regimesinvolve administration once per every two weeks or once a month or onceevery 3 to 6 months. Exemplary dosage schedules include 1-10 mg/kg or 15mg/kg on consecutive days, 30 mg/kg on alternate days or 60 mg/kgweekly. In some methods, two or more monoclonal antibodies withdifferent binding specificities are administered simultaneously, inwhich case the dosage of each antibody administered falls within theranges indicated.

Antibody is usually administered on multiple occasions. Intervalsbetween single dosages can be weekly, monthly or yearly. Intervals canalso be irregular as indicated by measuring blood levels of antibody toAβ in the patient. In some methods, dosage is adjusted to achieve aplasma antibody concentration of 1-1000 μg/ml and in some methods 25-300μg/ml. Alternatively, antibody can be administered as a sustainedrelease formulation, in which case less frequent administration isrequired. Dosage and frequency vary depending on the half-life of theantibody in the patient. In general, humanized antibodies show thelongest half-life, followed by chimeric antibodies and nonhumanantibodies.

The dosage and frequency of administration can vary depending on whetherthe treatment is prophylactic or therapeutic. In prophylacticapplications, compositions containing the present antibodies or acocktail thereof are administered to a patient not already in thedisease state to enhance the patient's resistance. Such an amount isdefined to be a “prophylactic effective dose.” In this use, the preciseamounts again depend upon the patient's state of health and generalimmunity, but generally range from 0.1 to 25 mg per dose, especially 0.5to 2.5 mg per dose. A relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives.

In therapeutic applications, a relatively high dosage (e.g., from about1 to 200 mg of antibody per dose, with dosages of from 5 to 25 mg beingmore commonly used) at relatively short intervals is sometimes requireduntil progression of the disease is reduced or terminated, andpreferably until the patient shows partial or complete amelioration ofsymptoms of disease. Thereafter, the patent can be administered aprophylactic regime.

Doses for nucleic acids encoding antibodies range from about 10 ng to 1g, 100 ng to 100 mg, 1 μg to 10 mg, or 30-300 μg DNA per patient. Dosesfor infectious viral vectors vary from 10-100, or more, virions perdose.

Therapeutic agents can be administered by parenteral, topical,intravenous, oral, subcutaneous, intraarterial, intracranial,intraperitoneal, intranasal or intramuscular means for prophylacticand/or therapeutic treatment. The most typical route of administrationof an immunogenic agent is subcutaneous although other routes can beequally effective. The next most common route is intramuscularinjection. This type of injection is most typically performed in the armor leg muscles. In some methods, agents are injected directly into aparticular tissue where deposits have accumulated, for exampleintracranial injection. Intramuscular injection or intravenous infusionare preferred for administration of antibody. In some methods,particular therapeutic antibodies are injected directly into thecranium. In some methods, antibodies are administered as a sustainedrelease composition or device, such as a Medipad™ device.

Agents of the invention can optionally be administered in combinationwith other agents that are at least partly effective in treatment ofamyloidogenic disease. In certain embodiments, a humanized antibody ofthe invention (e.g., humanized 12A11) is administered in combinationwith a second immunogenic or immunologic agent. For example, a humanized12A11 antibody of the invention can be administered in combination withanother humanized antibody to Aβ. In other embodiments, a humanized12A11 antibody is administered to a patient who has received or isreceiving an Aβ vaccine. In the case of Alzheimer's and Down's syndrome,in which amyloid deposits occur in the brain, agents of the inventioncan also be administered in conjunction with other agents that increasepassage of the agents of the invention across the blood-brain barrier.Agents of the invention can also be administered in combination withother agents that enhance access of the therapeutic agent to a targetcell or tissue, for example, liposomes and the like. Coadministeringsuch agents can decrease the dosage of a therapeutic agent (e.g.,therapeutic antibody or antibody chain) needed to achieve a desiredeffect.

C. Pharmaceutical Compositions

Agents of the invention are often administered as pharmaceuticalcompositions comprising an active therapeutic agent, i.e., and a varietyof other pharmaceutically acceptable components. See Remington'sPharmaceutical Science (15th ed., Mack Publishing Company, Easton, Pa.(1980)). The preferred form depends on the intended mode ofadministration and therapeutic application. The compositions can alsoinclude, depending on the formulation desired,pharmaceutically-acceptable, non-toxic carriers or diluents, which aredefined as vehicles commonly used to formulate pharmaceuticalcompositions for animal or human administration. The diluent is selectedso as not to affect the biological activity of the combination. Examplesof such diluents are distilled water, physiological phosphate-bufferedsaline, Ringer's solutions, dextrose solution, and Hank's solution. Inaddition, the pharmaceutical composition or formulation may also includeother carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenicstabilizers and the like.

Pharmaceutical compositions can also include large, slowly metabolizedmacromolecules such as proteins, polysaccharides such as chitosan,polylactic acids, polyglycolic acids and copolymers (such as latexfunctionalized Sepharose™, agarose, cellulose, and the like), polymericamino acids, amino acid copolymers, and lipid aggregates (such as oildroplets or liposomes). Additionally, these carriers can function asimmunostimulating agents (i.e., adjuvants).

For parenteral administration, agents of the invention can beadministered as injectable dosages of a solution or suspension of thesubstance in a physiologically acceptable diluent with a pharmaceuticalcarrier that can be a sterile liquid such as water oils, saline,glycerol, or ethanol. Additionally, auxiliary substances, such aswetting or emulsifying agents, surfactants, pH buffering substances andthe like can be present in compositions. Other components ofpharmaceutical compositions are those of petroleum, animal, vegetable,or synthetic origin, for example, peanut oil, soybean oil, and mineraloil. In general, glycols such as propylene glycol or polyethylene glycolare preferred liquid carriers, particularly for injectable solutions.Antibodies can be administered in the form of a depot injection orimplant preparation, which can be formulated in such a manner as topermit a sustained release of the active ingredient. An exemplarycomposition comprises monoclonal antibody at 5 mg/mL, formulated inaqueous buffer consisting of 50 mM L-histidine, 150 mM NaCl, adjusted topH 6.0 with HCl.

Typically, compositions are prepared as injectables, either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid vehicles prior to injection can also be prepared.The preparation also can be emulsified or encapsulated in liposomes ormicro particles such as polylactide, polyglycolide, or copolymer forenhanced adjuvant effect, as discussed above (see Langer, Science 249:1527 (1990) and Hanes, Advanced Drug Delivery Reviews 28:97 (1997)). Theagents of this invention can be administered in the form of a depotinjection or implant preparation, which can be formulated in such amanner as to permit a sustained or pulsatile release of the activeingredient.

Additional formulations suitable for other modes of administrationinclude oral, intranasal, and pulmonary formulations, suppositories, andtransdermal applications. For suppositories, binders and carriersinclude, for example, polyalkylene glycols or triglycerides; suchsuppositories can be formed from mixtures containing the activeingredient in the range of 0.5% to 10%, preferably 1%-2%. Oralformulations include excipients, such as pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, and magnesium carbonate. These compositions take the form ofsolutions, suspensions, tablets, pills, capsules, sustained releaseformulations or powders and contain 10%-95% of active ingredient,preferably 25%-70%.

Topical application can result in transdermal or intradermal delivery.Topical administration can be facilitated by co-administration of theagent with cholera toxin or detoxified derivatives or subunits thereofor other similar bacterial toxins (See Glenn et al., Nature 391, 851(1998)). Co-administration can be achieved by using the components as amixture or as linked molecules obtained by chemical crosslinking orexpression as a fusion protein.

Alternatively, transdermal delivery can be achieved using a skin patchor using transferosomes (Paul et al., Eur. J. Immunol. 25:3521 (1995);Cevc et al., Biochem. Biophys. Acta 1368:201-15 (1998)).

D. Monitoring the Course of Treatment

The invention provides methods of monitoring treatment in a patientsuffering from or susceptible to Alzheimer's, i.e., for monitoring acourse of treatment being administered to a patient. The methods can beused to monitor both therapeutic treatment on symptomatic patients andprophylactic treatment on asymptomatic patients. In particular, themethods are useful for monitoring passive immunization (e.g., measuringlevel of administered antibody).

Some methods involve determining a baseline value, for example, of anantibody level or profile in a patient, before administering a dosage ofagent, and comparing this with a value for the profile or level aftertreatment. A significant increase (i.e., greater than the typical marginof experimental error in repeat measurements of the same sample,expressed as one standard deviation from the mean of such measurements)in value of the level or profile signals a positive treatment outcome(i.e., that administration of the agent has achieved a desiredresponse). If the value for immune response does not changesignificantly, or decreases, a negative treatment outcome is indicated.

In other methods, a control value (i.e., a mean and standard deviation)of level or profile is determined for a control population. Typicallythe individuals in the control population have not received priortreatment. Measured values of the level or profile in a patient afteradministering a therapeutic agent are then compared with the controlvalue. A significant increase relative to the control value (e.g.,greater than one standard deviation from the mean) signals a positive orsufficient treatment outcome. A lack of significant increase or adecrease signals a negative or insufficient treatment outcome.Administration of agent is generally continued while the level isincreasing relative to the control value. As before, attainment of aplateau relative to control values is an indicator that theadministration of treatment can be discontinued or reduced in dosageand/or frequency.

In other methods, a control value of the level or profile (e.g., a meanand standard deviation) is determined from a control population ofindividuals who have undergone treatment with a therapeutic agent andwhose levels or profiles have plateaued in response to treatment.Measured values of levels or profiles in a patient are compared with thecontrol value. If the measured level in a patient is not significantlydifferent (e.g., more than one standard deviation) from the controlvalue, treatment can be discontinued. If the level in a patient issignificantly below the control value, continued administration of agentis warranted. If the level in the patient persists below the controlvalue, then a change in treatment may be indicated.

In other methods, a patient who is not presently receiving treatment buthas undergone a previous course of treatment is monitored for antibodylevels or profiles to determine whether a resumption of treatment isrequired. The measured level or profile in the patient can be comparedwith a value previously achieved in the patient after a previous courseof treatment. A significant decrease relative to the previousmeasurement (i.e., greater than a typical margin of error in repeatmeasurements of the same sample) is an indication that treatment can beresumed. Alternatively, the value measured in a patient can be comparedwith a control value (mean plus standard deviation) determined in apopulation of patients after undergoing a course of treatment.Alternatively, the measured value in a patient can be compared with acontrol value in populations of prophylactically treated patients whoremain free of symptoms of disease, or populations of therapeuticallytreated patients who show amelioration of disease characteristics. Inall of these cases, a significant decrease relative to the control level(i.e., more than a standard deviation) is an indicator that treatmentshould be resumed in a patient.

The tissue sample for analysis is typically blood, plasma, serum, mucousfluid or cerebrospinal fluid from the patient. The sample is analyzed,for example, for levels or profiles of antibodies to Aβ peptide, e.g.,levels or profiles of humanized antibodies. ELISA methods of detectingantibodies specific to Aβ are described in the Examples section. In somemethods, the level or profile of an administered antibody is determinedusing a clearing assay, for example, in an in vitro phagocytosis assay,as described herein. In such methods, a tissue sample from a patientbeing tested is contacted with amyloid deposits (e.g., from a PDAPPmouse) and phagocytic cells bearing Fc receptors. Subsequent clearing ofthe amyloid deposit is then monitored. The existence and extent ofclearing response provides an indication of the existence and level ofantibodies effective to clear Aβ in the tissue sample of the patientunder test.

The antibody profile following passive immunization typically shows animmediate peak in antibody concentration followed by an exponentialdecay. Without a further dosage, the decay approaches pretreatmentlevels within a period of days to months depending on the half-life ofthe antibody administered.

In some methods, a baseline measurement of antibody to Aβ in the patientis made before administration, a second measurement is made soonthereafter to determine the peak antibody level, and one or more furthermeasurements are made at intervals to monitor decay of antibody levels.When the level of antibody has declined to baseline or a predeterminedpercentage of the peak less baseline (e.g., 50%, 25% or 10%),administration of a further dosage of antibody is administered. In somemethods, peak or subsequent measured levels less background are comparedwith reference levels previously determined to constitute a beneficialprophylactic or therapeutic treatment regime in other patients. If themeasured antibody level is significantly less than a reference level(e.g., less than the mean minus one standard deviation of the referencevalue in population of patients benefiting from treatment)administration of an additional dosage of antibody is indicated.

Additional methods include monitoring, over the course of treatment, anyart-recognized physiologic symptom (e.g., physical or mental symptom)routinely relied on by researchers or physicians to diagnose or monitoramyloidogenic diseases (e.g., Alzheimer's disease). For example, one canmonitor cognitive impairment. The latter is a symptom of Alzheimer'sdisease and Down's syndrome but can also occur without othercharacteristics of either of these diseases. For example, cognitiveimpairment can be monitored by determining a patient's score on theMini-Mental State Exam in accordance with convention throughout thecourse of treatment.

E. Kits

The invention further provides kits for performing the monitoringmethods described above. Typically, such kits contain an agent thatspecifically binds to antibodies to Aβ. The kit can also include alabel. For detection of antibodies to Aβ, the label is typically in theform of labeled anti-idiotypic antibodies. For detection of antibodies,the agent can be supplied prebound to a solid phase, such as to thewells of a microtiter dish. Kits also typically contain labelingproviding directions for use of the kit. The labeling may also include achart or other correspondence regime correlating levels of measuredlabel with levels of antibodies to Aβ. The term labeling refers to anywritten or recorded material that is attached to, or otherwiseaccompanies a kit at any time during its manufacture, transport, sale oruse. For example, the term labeling encompasses advertising leaflets andbrochures, packaging materials, instructions, audio or videocassettes,computer discs, as well as writing imprinted directly on kits.

The invention also provides diagnostic kits, for example, research,detection and/or diagnostic kits (e.g., for performing in vivo imaging).Such kits typically contain an antibody for binding to an epitope of Aβ,preferably within residues 1-10. Preferably, the antibody is labeled ora secondary labeling reagent is included in the kit. Preferably, the kitis labeled with instructions for performing the intended application,for example, for performing an in vivo imaging assay. Exemplaryantibodies are those described herein.

F. In Vivo Imaging

The invention provides methods of in vivo imaging amyloid deposits in apatient. Such methods are useful to diagnose or confirm diagnosis ofAlzheimer's disease, or susceptibility thereto. For example, the methodscan be used on a patient presenting with symptoms of dementia. If thepatient has abnormal amyloid deposits, then the patient is likelysuffering from Alzheimer's disease. The methods can also be used onasymptomatic patients. Presence of abnormal deposits of amyloidindicates susceptibility to future symptomatic disease. The methods arealso useful for monitoring disease progression and/or response totreatment in patients who have been previously diagnosed withAlzheimer's disease.

The methods work by administering a reagent, such as antibody that bindsto Aβ, to the patient and then detecting the agent after it has bound.Preferred antibodies bind to Aβ deposits in a patient without binding tofull length APP polypeptide. Antibodies binding to an epitope of Aβwithin amino acids 1-10 are particularly preferred. In some methods, theantibody binds to an epitope within amino acids 7-10 of Aβ. Suchantibodies typically bind without inducing a substantial clearingresponse. In other methods, the antibody binds to an epitope withinamino acids 1-7 of Aβ. Such antibodies typically bind and induce aclearing response to Aβ. However, the clearing response can be avoidedby using antibody fragments lacking a full-length constant region, suchas Fabs. In some methods, the same antibody can serve as both atreatment and diagnostic reagent. In general, antibodies binding toepitopes C-terminal to residue 10 of Aβ do not show as strong a signalas antibodies binding to epitopes within residues 1-10, presumablybecause the C-terminal epitopes are inaccessible in amyloid deposits.Accordingly, such antibodies are less preferred.

Diagnostic reagents can be administered by intravenous injection intothe body of the patient, or directly into the brain by intracranialinjection or by drilling a hole through the skull. The dosage of reagentshould be within the same ranges as for treatment methods. Typically,the reagent is labeled, although in some methods, the primary reagentwith affinity for Aβ is unlabelled and a secondary labeling agent isused to bind to the primary reagent. The choice of label depends on themeans of detection. For example, a fluorescent label is suitable foroptical detection. Use of paramagnetic labels is suitable fortomographic detection without surgical intervention. Radioactive labelscan also be detected using PET or SPECT.

Diagnosis is performed by comparing the number, size, and/or intensityof labeled loci, to corresponding baseline values. The base line valuescan represent the mean levels in a population of undiseased individuals.Baseline values can also represent previous levels determined in thesame patient. For example, baseline values can be determined in apatient before beginning treatment, and measured values thereaftercompared with the baseline values. A decrease in values relative tobaseline signals a positive response to treatment.

The present invention will be more fully described by the followingnon-limiting examples.

EXAMPLES

The following Sequence identifiers are used throughout the Examplessection to refer to immunoglobulin chain variable region nucleotide andamino acid sequences. VL nucleotide VL amino acid VH nucleotide VH aminoacid Antibody sequence sequence sequence sequence 12A11 SEQ ID NO: 1 SEQID NO: 2 SEQ ID NO: 3 SEQ ID NO: 4 (coding) (coding) 12A11v1 SEQ ID NO:34 SEQ ID NO: 7 SEQ ID NO: 35 SEQ ID NO: 10 12A11v2 SEQ ID NO: 7 SEQ IDNO: 13 12A11v2.1 SEQ ID NO: 7 SEQ ID NO: 14 12A11v3 SEQ ID NO: 7 SEQ IDNO: 15 12A11 SEQ ID NO: 5 SEQ ID NO: 6

As used herein, an antibody or immunoglobulin sequence comprising a VLand/or VH sequence as set forth in any one of SEQ ID NOs: 1-4 cancomprise either the full sequence or can comprise the mature sequence(i.e., mature peptide without the signal or leader peptide).

Previous studies have shown that it is possible to predict in vivoefficacy of various Aβ antibodies in reducing AD-associatedneuropathology (e.g., plaque burden) by the ability of antibodies tobind plaques ex vivo (e.g., in PDAPP or AD brain sections) and/ortrigger plaque clearance in an ex vivo phagocytosis assay (Bard et al.(2000) Nat. Med. 6:916-919). The correlation supports the notion thatFc-dependent phagocytosis by microglial cells and/or macrophages isimportant to the process of plaque clearance in vivo. However, it hasalso been reported that antibody efficacy can also be obtained in vivoby mechanisms that are independent of Fc interactions (Bacskai et al.(2002) J. Neurosci. 22:7873-7878). Studies have indicated that anantibody directed against the midportion of Aβ, which cannot recognizeamyloid plaques, appears to bind to soluble Aβ and reduce plaquedeposition (DeMattos et al. (2001) Proc. Natl. Acad. Sci. USA98:8850-8855).

In order to characterize potential in vivo efficacy of the murinemonoclonal antibody 12A11 (isotype IgG1), various ex vivo assays werefirst performed.

Avidity of mAb 12A11 for Aβ-42. Binding of monoclonal antibody 12A11 toaggregated synthetic Aβ1-42 was performed by ELISA, as described inSchenk, et al. (Nature 400:173 (1999)). For comparison purposes, mAbs12B4, and 10D5 were also assayed. Soluble Aβ1-42 refers to the syntheticAβ1-42 peptide sonicated in dimethyl sulfoxide (DMSO). Serial dilutionsof the antibodies at 20 μg/ml were incubated with 50,000 cpm[¹²⁵I]Aβ1-42 (190 μCi/μmol; labeling with Iodogen reagent, Pierce)overnight at room temperature. Fifty microliters of a slurry containing75 mg/ml protein A Sepharose (Amersham Pharmacia) and 200 μg of rabbitanti-mouse IgG (H+L) (Jackson ImmunoResearch) were incubated with thediluted antibodies for 1 h at room temperature, washed twice, andcounted on a Wallac gamma counter (Perkin-Elmer). All steps wereperformed in RIA buffer consisting of 10 mM Tris, 0.5 M NaCl, 1 mg/mlgelatin, and 0.5% Nonidet P-40, pH 8.0.

Results from the avidity study are shown below in Table 2. TABLE 2 ED₅₀on aggregated % Capture of Antibody Epitope Isotype Aβ1-42, pM solubleAβ1-42 10D5^(†) Aβ3-7 IgG1  53 1 12B4^(†) Aβ3-7 IgG2a 667 8 12A11 Aβ3-7IgG1 233 30 ^(†)Antibodies 10D5 and 12B4 are described in WO 02/46237 andInternational Patent Application Serial No. PCT/US03/07715,respectively.*As a comparison, the anitbody 266 at 10 μg/ml would capture 70% ofAβ1-42.

All of the antibodies tested exhibited a high avidity for aggregatedAβ1-42. Moreover, antibodies 12B4 and 12A11 appreciably captured solubleAβ1-42 at antibody concentrations of 20 μg/ml. As shown in Table 2, theIgG1 antibody 12A11 captured Aβ1-42 more efficiently than the IgG2aantibody 12B4 or the IgG1 antibody 10D5.

The ability of various antibodies (including 12A11) to capture solubleAβ was further assayed as follows. Various concentrations of antibody(up to 10 μg/ml) were incubated with 50,000 CPM of ¹²⁵I-Aβ1-42 (or¹²⁵I-Aβ 1-40). The concentration of antibody sufficient to bind 25% ofthe radioactive counts was determined in a capture radioimmunoassay. Forantibodies not capable of binding 25% of the counts at 10 μg/ml, thepercentage of counts bound at 10 μg/ml was determined. The 12A11 bound20% of the radioactive counts (i.e., ¹²⁵I-Aβ at 10 μg/mil. This wasgreater than the amount bound by two other Aβ3-7 antibodies tested,namely 12B4 and 10D5 (binding 7% and 2% at 10 μg/ml, respectively).Thus, of the N-terminal (epitope Aβ3-7) antibodies tested, 12A11exhibited the most appreciable ability to capture Aβ.

As a measure of their ability to trigger Fc-mediated plaque clearance,the antibodies were also compared in an ex vivo phagocytosis assay withprimary mouse microglial cells and sections of brain tissue from PDAPPmice. Irrelevant IgG1 and IgG2a antibodies, having no reactivity towardAβ or other components of the assay, were used as isotype-matchednegative controls. Briefly, murine primary microglial cells werecultured with unfixed cryostat sections of PDAPP mouse brain in thepresence of antibodies. After 24 h of incubation, the total level of Aβremaining in the cultures was measured by ELISA. To quantify the degreeof plaque clearance/Aβ degradation, Aβ was extracted from the culturesof microglia and brain sections (n=3) with 8 M urea for ELISA analysis.Data were analyzed with ANOVA followed by a post hoc Dunnett's test.

As shown in FIG. 1, the 12B4 antibody reduced Aβ levels efficiently (73%for 12B4; P<0.001) with 12A11 showing somewhat less, albeitstatistically significant, efficiency (48% for 12A11, P<0.05). The 10D5antibody did not significantly reduce Aβ levels. The performance of12A11 in the ex vivo phagocytosis assay may be improved upon conversionto the IgG2a isotype which is a preferred isotype for microglialphagocytosis.

Example II In Vivo Efficacy of Mouse 12A11 Antibody

Mouse Antibody 12A11 Reduces Alzheimer 's-Like Neuropathology In Vivo Todetermine the in vivo efficacy of 12A11, antibodies (including 12A11,12B4, or 10D5) were administered to mice at 10 mg/kg by weeklyintraperitoneal injection for 6 months as described in Bard et al.(2000) Nat. Med. 6:916. At the end of the study, total levels ofcortical Aβ were determined by ELISA. As shown in FIG. 2A, each of theantibodies significantly reduced total Aβ levels compared with the PBScontrol (P<0.001), i.e. 12B4 showed a 69% reduction, 10D5 showed a 52%reduction, and 12A11 showed a 31% reduction.

The level of neuritic dystrophy was then examined in sections of braintissue from the above-mentioned mice to determine the associationbetween plaque clearance and neuronal protection. Data from brain imageanalyses examining the percentage of frontal cortex occupied by neuroticdystrophy is shown in FIG. 2B. These data show that antibodies 10D5 and12A11 were not effective at reducing neuritic dystrophy whereas 12B4significantly reduced neuritic dystrophy (12B4, P<0.05; ANOVA followedby post hoc Dunnett's test), as determined by the assay describedherein. Again, this activity of 12A11 may be improved by converting12A11 to the IgG2a isotype (murine efficacy). Regarding humanizedversions of 12A11, IgG1 isotypes are preferred for reducing neuriticdystrophy.

Experiments demonstrating the binding properties and in vivo efficacy ofantibody 12A11 are also described in Bard, et al. PNAS 100:2023 (2003),incorporated by reference herein.

In summary, all antibodies had significant avidity for aggregated Aβ andtriggered plaque clearance in an ex vivo assay. The IgG2a isotype(affinity for Fc receptors, in particular, FcγRI) appears to be animportant attribute for both clearance of Aβ and protection againstneuritic dystrophy. The antibody 12A11 (IgG1) captured soluble monomericAβ1-42 more efficiently than 12B4 (IgG2a) or 10D5 (IgG1) but was not aseffective at reducing neuritic dystrophy. Enhanced efficacy in reducingplaque burden and reducing neuritic dystrophy may be achieved byengineering antibodies to have an isotype which maximally supportsphagocytosis. Particularly efficacious antibodies bind to epitopeswithin the N terminus of Aβ.

Example III Cloning and Sequencing of the Mouse 12A11 Variable Regions

Cloning and Sequence Analysis of 12A11 VH. The VH and VL regions of12A11 from hybridoma cells were cloned by RT-PCR and 5′ RACE using mRNAfrom hybridoma cells and standard cloning methodology. The nucleotidesequence (coding, SEQ ID NO:3) and deduced amino acid sequence (SEQ IDNO:4) derived from independent cDNA clones encoding the presumed 12A11VH domain, are set forth in Table 3 and Table 4, respectively. TABLE 3Mouse 12A11 VH DNA sequence.ATGGACAGGCTTACTACTTCATTCCTGCTGCTGATTGTCCCTGCATATGTCTT (SEQ ID NO:3)GTCCCAAGTTACTCTAAAAGAGTCTGGCCCTGGGATATTGAAGCCCTCACAGACCCTCAGTCTGACTTGTTCTTTCTCTGGGTTTTCACTGAGCACTTCTGGTATGAGTGTAGGCTGGATTCGTCAGCCTTCAGGGAAGGGTCTGGAGTGGCTGGCACACATTTGGTGGGATGATGATAAGTACTATAACCCATCCCTGAAGAGCCGGCTCACAATCTCCAAGGATACCTCCAGAAACCAGGTATTCCTCAAGATCACCAGTGTGGACACTGCAGATACTGCCACTTACTACTGTGCTCGAAGAACTACTACGGCTGACTACTTTGCCTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA

TABLE 4 Mouse 12A11 VH amino acid sequencemdrlttsflllivpayvlsQVTLKESGPGILKPSQTLSLTCSFSGFSLStsgmsvgWIRQPSGKG (SEQID NO: 4) LEWLAhiwwdddkyynpslksRLTISKDTSRNQVFLKITSVDTADTATYYCARrtttadyfayWGQGTTLTVSS* Leader peptide and CDRs in lower case.

Cloning and Sequence Analysis of 12 μl VL. The light chain variable VLregion of 12A11 was cloned in an analogous manner as the VH region. Thenucleotide sequence (coding, SEQ ID NO:1) and deduced amino acidsequence (SEQ ID NO:2) derived from two independent cDNA clones encodingthe presumed 12A11 VL domain, are set forth in Table 5 and Table 6,respectively. TABLE 5 Mouse 12A11 VL DNA sequenceATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTCCAG (SEQ ID NO:1)CAGTGATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTGTACATAGTAATGGAAACACCTACTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAATTTATTACTGCTTTCAAAGTTCACATGTTCCTCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA

TABLE 6 Mouse 12A11 VL amino acid sequencemklpvrllvlmfwipasssDVLMTQTPLSLPVSLGDQASISCrssqsivhsngntyleWYLQKPGQ (SEQID NO: 2) SPKLLIYkvsnrfsGVPDRFSGSGSGTDFTLKISRVEAEDLGIYYCfqsshvpltFGAGTKLELK* Leader peptide and CDRs in lower case.

The 12A11 VL and VH sequences meet the criteria for functional V regionsin so far as they contain a contiguous ORF from the initiator methionineto the C-region, and share conserved residues characteristic ofimmunoglobulin V region genes. From N-terminal to C-terminal, both lightand heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3and FR4.

Example IV Expression of Chimeric 12A11 Antibody

Expression of Chimeric 12 μl Antibody: The variable heavy and lightchain regions were re-engineered to encode splice donor sequencesdownstream of the respective VDJ or VJ junctions, and cloned into themammalian expression vector pCMV-hγl for the heavy chain, and pCMV-hκlfor the light chain. These vectors encode human γl and Ck constantregions as exonic fragments downstream of the inserted variable regioncassette. Following sequence verification, the heavy chain and lightchain expression vectors were co-transfected into COS cells. Variousheavy chain clones were independently co-transfected with differentchimeric light chain clones to confirm reproducibility of the result.Antibodies were immunoprecipitated from COS cell conditioned media usingprotein A Sepharose. Antibody chains were detected on immunoblots ofSDS-PAGE gels. Detection was accomplished using goat-anti-human-IgG(H+L) antibody at a 1:5000 dilution at room temperature for 1 hour.Significant quantities of 12A11H+L chain were detected in conditionedmedia.

Direct binding of chimeric 12A11 antibody to Aβ was tested by ELISAassay. FIG. 4 demonstrates that chimeric 12A11 was found to bind to Aβwith high avidity, similar to that demonstrated by chimeric andhumanized 3D6. (The cloning, characterization and humanization of 3D6 isdescribed in U.S. patent application Ser. No. 10/010,942, the entirecontent of which is incorporated herein by this reference.) Bindingavidity was also similar to that demonstrated by chimeric and humanized12B4. (The cloning, characterization and humanization of 12B4 isdescribed in U.S. patent application Ser. No. 10/388,214, the entirecontent of which is incorporated herein by this reference.)

Example V 12A11 Humanization

A. 12A11 Humanized Antibody, Version 1

Homology/Molecular Model Analysis. In order to identify key structuralframework residues in the murine 12A11 antibody, three-dimensionalmodels were studied for solved murine antibodies having homology to the12A11 heavy and light chains. An antibody designated 1KTR was chosenhaving close homology to the 12A11 light chain and two antibodiesdesignated 1ETZ and 1JRH were chosen having close homology to the 12A11heavy chain. These mouse antibodies show strong sequence conservationwith 12A11 (94% identity in 112 amino acids for Vk and 83% identity in126 amino acids and 86% identity in 121 amino acids respectively forVh). The heavy chain structure of 1ETZ was superimposed onto that of1KTR. In addition, for Vk the CDR loops of the selected antibody fallinto the same canonical Chothia structural classes as do the CDR loopsof 12A11 VL. The crystal structures of these antibodies were exeminedfor residues (e.g., FR residues important for CDR conformation, etc.)predicted be important for function of the antibody, and by comparison,function of the similar 12A11 antibody.

Selection of Human Acceptor Antibody Sequences. Suitable human acceptorantibody sequences were identified by computer comparisons of the aminoacid sequences of the mouse variable regions with the sequences of knownhuman antibodies. The comparison was performed separately for the 12A11heavy and light chains. In particular, variable domains from humanantibodies whose framework sequences exhibited a high degree of sequenceidentity with the murine VL and VH framework regions were identified byquery of the NCBI Ig Database using NCBI BLAST (publicly accessiblethrough the National Institutes of Health NCBI internet server) with therespective murine framework sequences.

Two candidate sequences were chosen as acceptor sequences based on thefollowing criteria: (1) homology with the subject sequence; (2) sharingcanonical CDR structures with the donor sequence; and/or (3) notcontaining any rare amino acid residues in the framework regions. Theselected acceptor sequence for VL is BAC01733 in the NCBI Ignon-redundant database. The selected acceptor sequence for VH isAAA69734 in the NCBI Ig non-redundant database. AAA69734 is a humansubgroup III antibody (rather than subgroup II) but was selected as aninitial acceptor antibody based at least in part on the reasoning inSaldanha et al. (1999) Mol. Immunol. 36:709. First versions of humanized12A11 antibody utilize these selected acceptor antibody sequences. Theantibody is described in Schroeder and Wang (1990) Proc. Natl. Acad.Sci. USA 872:6146.

Substitution of Amino Acid Residues. As noted supra, the humanizedantibodies of the invention comprise variable framework regionssubstantially from a human immunoglobulin (acceptor immunoglobulin) andcomplementarity determining regions substantially from a mouseimmunoglobulin (donor immunoglobulin) termed 12A11. Having identifiedthe complementarity determining regions of 12A11 and appropriate humanacceptor immunoglobulins, the next step was to determine which, if any,residues from these components to substitute to optimize the propertiesof the resulting humanized antibody.

Reshaped Light Chain V Region:

The amino acid alignment of the reshaped light chain V region is shownin FIG. 5A. The choice of the acceptor framework (BAC01733) is from thesame human subgroup as that which corresponds to the murine V region,has no unusual framework residues, and the CDRs belong to the sameChothia canonical structure groups. No backmutations were made inVersion 1 of humanized 12A11.

Reshaped Heavy Chain V Region:

The amino acid alignment of the reshaped heavy chain V region is shownin FIG. 5B. The choice for the acceptor framework (AAA69734) is fromhuman subgroup III (as described previously) and has no unusualframework residues. Structural analysis of the murine VH chain (1ETZ and1JRH), in conjunction with the amino acid alignment of AAA69734 to themurine sequence dictates 9 backmutations in version 1 (v1) of thereshaped heavy chain: A24F T28S F29L V37I V48L F67L R71K N73T L78V(Kabat numbering). The back mutations are highlighted by asterisks inthe amino-acid alignment shown in FIG. 5B.

Of the 9 back mutations, 3 are dictated by the model because theresidues are canonical residues (A24F, F29L, & R71K, solid fill), i.e.framework residues which may contribute to antigen binding by virtue ofproximity to CDR residues. There is one back mutation in the next mostimportant class of residues, the interface residues involved in VH-VLpacking interactions (underlined), i.e., V37I. The N73T mutation is at avernier residue (dotted fill) on the edge of the binding site, possiblyinteracting with S30 adjacent to CDR1. The remaining 4 residues targetedfor back mutation (T28S, V48L, F67L, L78V, Kabat numbering) also fallinto the vernier class (indirect contribution to CDR conformation,dotted fill in FIG. 5B).

A summary of the changes incorporated into version 1 of humanized 12A11is presented in Table 7. TABLE 7 Summary of changes in humanized12A11.v1 Changes VL (112 residues) VH (120 residues) Hu->Mu:  0/112 9/120 Framework CDR1  5/16  6/7 CDR2  3/7 10/16 CDR3  6/8  8/11 TotalHu->Mu 14/112 (12.5%) 33/120 (27.5%) Mu->Hu: 11/112 26/120 FrameworkBackmutation none 1. Canonical: A24F, F29L, notes   R71K 2. Packing:V37I 3. Vernier: T28S, V48L,   F67L, N73T, L78V Acceptor 4. Genbank Acc.no. 7. Genbank Acc. no. notes   BAC01733   AAA69734 (H1 - 5. CDRs fromsame   class 1, H2 = class 3)   canonical structural 8. CDRs from same  group as donor mouse;   canonical structural 6. Immunoglobulin kappa  group as donor mouse   light chain K64(AIMS4) 9. fetal Ig

Tables 8 and 9 set forth Kabat numbering keys for the various light andheavy chains, respectively. TABLE 8 Key to Kabat Numbering for 12A11Light Chain mouse HUM A19- KAB 12A11 12A11 BAC Germ- # # TYPE VL VL01733 line Comment  1 1 FR1 D D D D  2 2 V V V I canonical  3 3 L V V V 4 4 M M M M vernier  5 5 T T T T  6 6 Q Q Q Q  7 7 T S S S  8 8 P P P P 9 9 L L L L  10 10 S S S S  11 11 L L L L  12 12 P P P P  13 13 V V V V 14 14 S T T T  15 15 L P P P  16 16 G G G G  17 17 D E E E  18 18 Q P PP  19 19 A A A A  20 20 S S S S  21 21 I I I I  22 22 S S S S  23 23 C CC C  24 24 CDR1 R R R R  25 25 S S S S  26 26 S S S S  27 27 Q Q Q Q 27A 28 S S S S  27B 29 I I L L  27C 30 V V L L  27D 31 H H H H  27E 32S S S S  28 33 N N N N  29 34 G G G G  30 35 N N Y Y  31 36 T T N N  3237 Y Y Y Y  33 38 L L L L  34 39 E E D D  35 40 FR2 W W W W  36 41 Y Y YY packing  37 42 L L L L  38 43 Q Q Q Q packing  39 44 K K K K  40 45 PP P P vernier  41 46 G G G G  42 47 Q Q Q Q  43 48 S S S S  44 49 P P PP packing  45 50 K Q Q Q  46 51 L L L L packing  47 52 L L L L vernier 48 53 I I I I canonical  49 54 Y Y Y Y vernier  50 55 CDR2 K K L L  5156 V V G G  52 57 S S S S  53 58 N N N N  54 59 R R R R  55 60 F F A A 56 61 S S S S  57 62 FR3 G G G G  58 63 V V V V  59 64 P P P P  60 65 DD D D  61 66 R R R R  62 67 F F F F  63 68 S S S S  64 69 G G G Gcanonical  65 70 S S S S  66 71 G G G G vernier  67 72 S S S S  68 73 GG G G vernier  69 74 T T T T vernier  70 75 D D D D  71 76 F F F Fcanonical  72 77 T T T T  73 78 L L L L  74 79 K K K K  75 80 I I I I 76 81 S S S S  77 82 R R R R  78 83 V V V V  79 84 E E E E  80 85 A A AA  81 86 E E E E  82 87 D D D D  83 88 L V V V  84 89 G G G G  85 90 I VV V  86 91 Y Y Y Y  87 92 Y Y Y Y packing  88 93 C C C C  89 94 CDR3 F FM M  90 95 Q Q Q Q  91 96 S S A A  92 97 S S L L  93 98 H H Q Q  94 99 VV T T  95 100 P P P P  96 101 L L Y  97 102 T T T  98 103 FR4 F F Fpacking  99 104 G G G 100 105 A Q Q 101 106 G G G 102 107 T T T 103 108K K K 104 109 L L L 105 110 E E E 106 111 L I I 106A 112 K K K

TABLE 9 Key to Kabat Numbering for 12A11 Heavy Chain Mouse HUM 12A1112A11 AAA 567123 KAB # # TYPE VH VHv1 69734 Germline Comment  1 1 FR1 QQ Q Q  2 2 V V V V vernier  3 3 T Q Q Q  4 4 L L L L  5 5 K V V V  6 6 EE E E  7 7 S S S S  8 8 G G G G  9 9 P G G G 10 10 G G G G 11 11 I V V V12 12 L V V V 13 13 K Q Q Q 14 14 P P P P 15 15 S G G G 16 16 Q R R R 1717 T S S S 18 18 L L L L 19 19 S R R R 20 20 L L L L 21 21 T S S S 22 22C C C C 23 23 S A A A 24 24 F F A A canonical for H1- backmutate in v125 25 S S S S 26 26 G G G G canonical 27 27 F F F F canonical 28 28 S ST T vernier, close to H1- backmutate in v1 29 29 L L F F canonical forH1- backmutate in v1 30 30 S S S S 31 31 CDR1 T T S S 32 32 S S Y Y 3333 G G A A 34 34 M M M M 35 35 S S H H  35A 36 V V — —  35B 37 G G — —36 38 FR2 W W W W 37 39 I I V V packing - bacmutate in v1 38 40 R R R R39 41 Q Q Q Q packing 40 42 P A A A 41 43 S P P P 42 44 G G G G 43 45 KK K K 44 46 G G G G 45 47 L L L L packing 46 48 E E E E 47 49 W W W Wpacking 48 50 L L V V vernier (underneath H2) - backmutate in v1 49 51 AA A A 50 52 CDR2 H H V V 51 53 I I I I 52 54 W W S S 53 55 W W Y Y 54 56D D D D 55 57 D D G G — — S S 56 58 D D N N 57 59 K K K K 58 60 Y Y Y Y59 61 Y Y Y Y 60 62 N N A A 61 63 P P D D 62 64 S S S S 63 65 L L V V 6466 K K K K 65 67 S S G G 66 68 FR3 R R R R 67 69 L L F F vernier(underneath H2, possibly interacting with L63) - backmutate in v1 68 70T T T T 69 71 I I I I 70 72 S S S S 71 73 K K R R canonical for H2 -backmutate in v1 72 74 D D D D 73 75 T T N N vernier (edge of bindingsite, possibly interacting with S30) - backmutate in v1 74 76 S S S S 7577 R K K K 76 78 N N N N 77 79 Q T T T 78 80 V V L L vernier (buriedunder H1, possibly interacting with V35A) - backmutate in v1 79 81 F Y YY 80 82 L L L L 81 83 K Q Q Q 82 84 I M M M  82A 85 T N N N  82B 86 S SS S  82C 87 V L L L 83 88 D R R R 84 89 T A A A 85 90 A E E E 86 91 D DD D 87 92 T T T T 88 93 A A A A 89 94 T V V V 90 95 Y Y Y Y 91 96 Y Y YY packing 92 97 C C C C 93 98 A A A A packing 94 99 R R R R canonical —— D D 95 100 CDR3 R R R — 96 101 T T H — 97 102 T T S — 98 103 T T S —99 104 A A S A 100  105 D D W K 100A 106 Y Y Y L 100B 107 F F Y L 101 108 A A G M 102  109 Y Y M L — — D L — — V I 103  110 W W W S packing104  111 G G G G 105  112 Q Q Q A 106  113 G G G K 107  114 FR4 T T T G108  115 T T T Q 109  116 L V V W 110  117 T T T S 111  118 V V V P 112 119 S S S S 113  120 S S S L

The humanized antibodies preferably exhibit a specific binding affinityfor A βof at least 10⁷, 10⁸, 10⁹ or 10¹⁰ M⁻¹. Usually the upper limit ofbinding affinity of the humanized antibodies for Aβ is within a factorof three, four or five of that of 12A11 (i.e., ˜10⁹ M⁻¹). Often thelower limit of binding affinity is also within a factor of three, fouror five of that of 12A11.

Assembly and Expression of Humanized 12A11 VH and VL, Version 1

PCR-mediated assembly was used to generate h12A11 v1 using appropriateoligonucleotide primers. The nucleotide sequences of humanized 12A11VL(version 1) (SEQ ID NO:34) and 12A11VH (version 1) (SEQ ID NO: 35) arelisted below in Tables 10 and 11, respectively. TABLE 10 Nucleotidesequence of humanized 12A11VLv1.atgaggctccctgctcagctcctggggctgctgatgctctgggtctctggctccagtgggGATGTTGTGAT(SEQ ID NO:34)GACCCAATCTCCACTCTCCCTGCCTGTCACTCCTGGAGAGCCAGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTGTGCATAGTAATGGAAACACCTACCTGGAATGGTACCTGCAGAAACCAGGCCAGTCTCCACAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATGTGGGAGTTTATTACTGCTTTCAAAGTTCACATGTTCCTCTCACCTTCGGTCAGGGGACCAAGCTGGAGATCAAA(uppercase VL segment only) leader peptide encoded by A19 germline seqderived from x63397 leader peptide

TABLE 11 Nucleotide sequence of humanized 12A11VHv1atggagtttgggctgagctgggttttcctcgttgctcttctgagaggtgtccagtgtCAAGTTCAGCTGGT(SEQ ID NO:35)GGAGTCTGGCGGCGGGGTGGTGCAGCCCGGACGGTCCCTCAGGCTGTCTTGTGCTTTCTCTGGGTTTTCACTGAGCACTTCTGGTATGAGTGTGGGCTGGATTCGTCAGGCTCCAGGGAAGGGTCTGGAGTGGCTGGCACACATTTGGTGGGATGATGATAAGTACTATAACCCATCCCTGAAGAGCCGGCTCACAATCTCCAAGGATACCTCCAAAAACACCGTGTACCTCCAGATGAACAGTCTGCGGGCTGAAGATACTGCCGTGTACTACTGTGCTCGAAGAACTACTACCGCTGACTACTTTGCCTACTGGGGCCAAGGCACCACTGTCACAGTCTCCTCALeader peptide (lower case) derived from VH donor seq accessionM34030.1/aaa69734/M72

The amino acid sequences of humanized 12A11 VL (version 1) (SEQ ID NO:7)and 12A11 VH (version 1) (SEQ ID NO: 10) are depicted in FIGS. 5A and5B, respectively.

B. Humanized 12A11 Antibodies—Versions 2, 2.1 and 3

The vernier residues (e.g., S28T, V48L, F67L, L78V) contributeindirectly to CDR conformation and were postulated to be of leastsignificance for conformational perturbation. The targeted residues weremutated by site-directed mutagenesis using a kit by Strategene andh12A11 VHv1 in a pCRS plasmid as the mutagenesis template to arise atclones corresponding to version 2. A sequenced verified V-region insertof version 2 was subcloned into the BamHI/HindIII sites of the heavychain expression vector pCMV-Cgamma1 to produce recombinant h12A11v2antibody. A version 2.1 antibody was similarly created having each ofthe above venier residue mutations (i.e., elimination of backmutations)in addition to mutation at position T73N. A version 3 antibody likewisehad each of the above mutations, T28S, L48V, L67F, V78L, in addition toa mutation at position K71R.

C. Humanized 12A11 Antibodies—Versions 4 to 6

Additional humanized 12A11 versions were designed which retainedbackmutations at canonical and packing residues but eliminatedbackmutations at one (versions 4.1 to 4.4), two (versions 5.1 to 5.6) orthree (versions 6.1 to 6.4) vernier residues. Site-directed mutagenesisand clone construction was performed as described in subpart C, above.Recombinant antibodies were expresed in COS cells and purified from COScell supernatants. Additional versions may include combinations of theabove, for example, human residues at 1, 2, 3, 4 or 5 vernier residuesin combination with at least one packing and/or canonical residue (e.g.,human residues at positions 28, 37, 48, 67, 71 and 78 or human residuesat positions 28, 37, 48, 67, 71, 73 and 78).

D. Humanized 12A11 Antibodies—Versions 7 and 8

A seventh version of humanized 12A11 is created having each of thebackmutations indicated for version 1, except for the T→S backmutationat residue 28 (vernier), and the V→I backmutation at residue 37(packing). An eighth version of humanized 12A11 is created having eachof the backmutations indicated for version 1, except for the N→Tbackmutation at residue 73 (vernier). The amino acid sequences ofhumanized 12A11 version 7 and 8 heavy chains are set forth as SEQ IDNOs: 30 and 31 respectively.

As compared to version 1, version 7 contains only 7 backmutations. TheT28S backmutation is conservative and is eliminated in version 7 of theheavy chain. The backmutation at packing residue V37I is also eliminatedin version 7. As compared to version 1, version 7 contains only 8backmutations. In version 8, the N73T (vernier) backmutation iseliminated.

Additional versions may include combinations of the above, for example,human residues (e.g., elimination of backmutations) at 1, 2, 3, 4 (or 5)residues selected from positons 28, 48, 78 and 73, optionally incombination with elimination of backmutation at at least one packingresidue (e.g., position 37) and/or at least one canonical residue.

Example VI Functional Texting of Humanized 12A11 Antibodies

Humanized 12A11 version 1 was cloned as described in Example V.Humanized 12A11 was produced by transient expression in COS cells, andpurified according to art-recognized methodologies. The binding activityof the humanized antibody was first demonstrated by a qualitative ELISAassay (data not shown). Humanized 12A11 version 1 was further comparedto its murine and chimeric counterparts for two properties: antigenbinding (quantitative Aβ ELISA) and relative affinity. The bindingactivity of h12A11v1 was demonstrated in the quantitative Aβ ELISA andfound to be identical with murine and chimeric forms of 12A11 (see FIG.7).

The affinity of h12A11 v1 antibody was also compared with murine andchimeric 12A11 antibodies by a competitive Aβ ELISA. For the competitivebinding assay, a biotin conjugated recombinant mouse 12A11Cγ2a (isotypeswitched 12A11) was used. The binding activity of the biotinylatedm12A11Cγ2a for aggregate Aβ1-42 was confirmed by an ELISA assay usingstrepavidin-HRP as reporter. A direct comparison of Aβ binding by thetwo isoforms of 12A11 (Cγ1, Cγ2a), using HRP conjugated goat anti-mouseHRP as reporter, confirmed that the biotin conjugated recombinant12A11Cγ2a is comparable to the original Cγ1 mouse antibody.

Competition binding study employed the biotin conjugated ml 2A11Cγ2a ata fixed concentration and competing with a range of concentrations oftest antibodies as indicated in FIG. 8. FIG. 8 shows the result ofh12A11v1 competitive assay comparing h12A11v1 with chimeric and murineforms. The humanized 12A11v1 competed within 2×IC50 value with itsmurine and chimeric counterparts. This data is consistent with affinitydetermination using Biacore technology (data not shown), which indicatedKD values of 38 nM and 23 nM for the murine Cγ2a and h12A11v1,respectively. In summary, the findings suggest h12A11 v1 retains theantigen binding properties and affinity of its original murinecounterpart.

COS cells were transiently transfected with different combinations ofhumanized 12A11VH and h12A11VLv1. The conditioned media was collected 72hours post-transfection. Antibody concentration in conditioned mediafrom transfected COS cells was determined by a quantitative human IgGELISA. Quantitative Aβ (1-42) aggregate binding assay confirmed thath12A11v2, v2.1 and v3 are comparable to h12A11v1 and to chimeric 12A11for antigen binding. Moreover, versions 5.1-5.6 and 6.1-6.3 exhibitsimilar binding activities when tested in this binding assay. Version6.4 showed some loss of activity in the assay but activity was notablyrestored in v2.

Binding properties for murine 12A11 and h12A11v1 were also comparedusing BIAcore technology. Murine 12A11 and h12A11v1 exhibited similarbinding profiles when exposed to either low- or high-density immobilizedAβ peptide (bio-DAE peptide). Kinetic analysis of murine 12A11 versush12A11v1 was also performed. In these studies the BIAcore technology wasused to measure the binding of soluble antibody to solid phase boundbiotinylated DAE peptide. The peptide was immobilized on streptavidinbiosensor chips then, varying concentrations of each antibody wereapplied in triplicates and the binding was measured as a function oftime. The kinetic data was analyzed using BIAevaluation software appliedto a bivalent model. The apparent dissociation (k_(d)) and association(k_(a)) rate constants were calculated from the appropriate regions ofthe sensorgrams using a global analysis. The affinity constant of theinteraction between bio-DAE10 and the antibodies was calculated from thekinetic rate constants. From these measurements the apparentdissociation (kd) and association (ka) rate constants were derived andused to calculate a K_(D) value for the interaction. Table 12 includes asummary of kinetic analysis of Aβ binding of 12A11 antibodies asdetermined by BIAcore analysis. TABLE 12 Bivalent Model (globalanalysis) Antibody Ka (1/Ms) Kd (1/s) KA (1/M) KD (nM) Chi2 m12A111.05E+05 3.98E−03 2.64E+07 38.00 0.247 h12A11v1 1.47E+05 3.43E−034.29E+07 23.30 0.145

The data indicate that humanized 12A11v1 has a similar affinity for Aβpeptide when compare with parental murine 12A11.

Example VII Prevention and Treatment of Human Subjects

A single-dose phase I trial is performed to determine safety in humans.A therapeutic agent is administered in increasing dosages to differentpatients starting from about 0.01 the level of presumed efficacy, andincreasing by a factor of three until a level of about 10 times theeffective mouse dosage is reached.

A phase II trial is performed to determine therapeutic efficacy.Patients with early to mid Alzheimer's Disease defined using Alzheimer'sdisease and Related Disorders Association (ADRDA) criteria for probableAD are selected. Suitable patients score in the 12-26 range on theMini-Mental State Exam (MMSE). Other selection criteria are thatpatients are likely to survive the duration of the study and lackcomplicating issues such as use of concomitant medications that mayinterfere. Baseline evaluations of patient function are made usingclassic psychometric measures, such as the MMSE, and the ADAS, which isa comprehensive scale for evaluating patients with Alzheimer's Diseasestatus and function. These psychometric scales provide a measure ofprogression of the Alzheimer's condition. Suitable qualitative lifescales can also be used to monitor treatment. Disease progression canalso be monitored by MRI. Blood profiles of patients can also bemonitored including assays of immunogen-specific antibodies and T-cellsresponses.

Following baseline measurements, patients begin receiving treatment.They are randomized and treated with either therapeutic agent or placeboin a blinded fashion. Patients are monitored at least every six months.Efficacy is determined by a significant reduction in progression of atreatment group relative to a placebo group.

A second phase II trial is performed to evaluate conversion of patientsfrom non-Alzheimer's Disease early memory loss, sometimes referred to asage-associated memory impairment (AAMI) or mild cognitive impairment(MCI), to probable Alzheimer's disease as defined as by ADRDA criteria.Patients with high risk for conversion to Alzheimer's Disease areselected from a non-clinical population by screening referencepopulations for early signs of memory loss or other difficultiesassociated with pre-Alzheimer's symptomatology, a family history ofAlzheimer's Disease, genetic risk factors, age, sex, and other featuresfound to predict high-risk for Alzheimer's Disease. Baseline scores onsuitable metrics including the MMSE and the ADAS together with othermetrics designed to evaluate a more normal population are collected.These patient populations are divided into suitable groups with placebocomparison against dosing alternatives with the agent. These patientpopulations are followed at intervals of about six months, and theendpoint for each patient is whether or not he or she converts toprobable Alzheimer's Disease as defined by ADRDA criteria at the end ofthe observation.

Although the foregoing invention has been described in detail forpurposes of clarity of understanding, it will be obvious that certainmodifications may be practiced within the scope of the appended claims.All publications and patent documents cited herein, as well as textappearing in the figures and sequence listing, are hereby incorporatedby reference in their entirety for all purposes to the same extent as ifeach were so individually denoted.

From the foregoing it will be apparent that the invention provides for anumber of uses. For example, the invention provides for the use of anyof the antibodies to Aβ described above in the treatment, prophylaxis ordiagnosis of amyloidogenic disease, or in the manufacture of amedicament or diagnostic composition for use in the same.

1. A humanized immunoglobulin light chain comprising (i) variable regioncomplementarity determining regions (CDRs) from the 12A11 immunoglobulinlight chain variable region sequence set forth as SEQ ID NO:2, and (ii)a variable framework region from a human acceptor immunoglobulin lightchain sequence.
 2. A humanized immunoglobulin light chain comprising (i)variable region complementarity determining regions (CDRs) from the12A11 immunoglobulin light chain variable region sequence set forth asSEQ ID NO:2, and (ii) a variable framework region from a human acceptorimmunoglobulin light chain sequence, provided that at least oneframework residue is substituted with the corresponding amino acidresidue from the mouse 12A11 light chain variable region sequence,wherein the framework residue is selected from the group consisting of:(a) a residue that non-covalently binds antigen directly; (b) a residueadjacent to a CDR; (c) a CDR-interacting residue; and (d) a residueparticipating in the VL-VH interface.
 3. A humanized immunoglobulinheavy chain comprising (i) variable region complementarity determiningregions (CDRs) from the 12A11 immunoglobulin heavy chain variable regionsequence set forth as SEQ ID NO:4, and (ii) a variable framework regionfrom a human acceptor immunoglobulin heavy chain
 4. A humanizedimmunoglobulin heavy chain comprising (i) variable regioncomplementarity determining regions (CDRs) from the 12A11 immunoglobulinheavy chain variable region sequence set forth as SEQ ID NO:4, and (ii)a variable framework region from a human acceptor immunoglobulin heavychain, provided that at least one framework residue is substituted withthe corresponding amino acid residue from the mouse 12A11 heavy chainvariable region sequence, wherein the framework residue is selected fromthe group consisting of (a) a residue that non-covalently binds antigendirectly; (b) a residue adjacent to a CDR; (c) a CDR-interactingresidue; and (d) a residue participating in the VL-VH interface.
 5. Thelight chain of claim 2, wherein a CDR-interacting residue is identifiedby modeling the 12A11 light chain based on the solved structure of amurine immunoglobulin light chain that shares at least 70% sequenceidentity with the 12A11 light chain.
 6. The light chain of claim 2,wherein a CDR-interacting residue is identified by modeling the 12A11light chain based on the solved structure of a murine immunoglobulinlight chain that shares at least 80% sequence identity with the 12A11light chain.
 7. The light chain of claim 2, wherein a CDR-interactingresidue is identified by modeling the 12A11 light chain based on thesolved structure of a murine immunoglobulin light chain that shares atleast 90% sequence identity with the 12A11 light chain.
 8. The heavychain of claim 4, wherein a CDR-interacting residue is identified bymodeling the 12A11 heavy chain based on the solved structure of a murineimmunoglobulin heavy chain that shares at least 70% sequence identitywith the 12A11 heavy chain.
 9. The heavy chain of claim 4, wherein aCDR-interacting residue is identified by modeling the 12A11 heavy chainbased on the solved structure of a murine immunoglobulin heavy chainthat shares at least 80% sequence identity with the 12A11 heavy chain.10. The heavy chain of claim 4, wherein a CDR-interacting residue isidentified by modeling the 12A11 heavy chain based on the solvedstructure of a murine immunoglobulin heavy chain that shares at least90% sequence identity with the 12A11 heavy chain.
 11. A humanizedimmunoglobulin light chain comprising (i) variable regioncomplementarity determining regions (CDRs) from the 12A11 immunoglobulinlight chain variable region sequence set forth as SEQ ID NO:2, and (ii)a variable framework region from a human acceptor immunoglobulin lightchain sequence, provided that at least one framework residue issubstituted with the corresponding amino acid residue from the mouse12A11 light chain variable region sequence, wherein the frameworkresidue is a residue capable of affecting light chain variable regionconformation or function as identified by analysis of athree-dimensional model of the 12A11 immunoglobulin light chain variableregion.
 12. A humanized immunoglobulin heavy chain comprising (i)variable region complementarity determining regions (CDRs) from the12A11 immunoglobulin heavy chain variable region sequence set forth asSEQ ID NO:4, and (ii) a variable framework region from a human acceptorimmunoglobulin heavy chain, provided that at least one framework residueis substituted with the corresponding amino acid residue from the mouse12A11 heavy chain variable region sequence, wherein the frameworkresidue is a residue capable of affecting heavy chain variable regionconformation or function as identified by analysis of athree-dimensional model of the 12A11 immunoglobulin heavy chain variableregion.
 13. The light chain of claim 11, wherein the framework residueis selected from the group consisting of a residue capable ofinteracting with antigen, a residue proximal to the antigen bindingsite, a residue capable of interacting with a CDR, a residue adjacent toa CDR, a residue within 6 Å of a CDR residue, a canonical residue, avernier zone residue, an interchain packing residue, a rare residue, anda glycoslyation site residue on the surface of the structural model. 14.The heavy chain of claim 12, wherein the framework residue is selectedfrom the group consisting of a residue capable of interacting withantigen, a residue proximal to the antigen binding site, a residuecapable of interacting with a CDR, a residue adjacent to a CDR, aresidue within 6 Å of a CDR residue, a canonical residue, a vernier zoneresidue, an interchain packing residue, an unusual residue, and aglycoslyation site residue on the surface of the structural model. 15.The light chain of claim 11, wherein the framework residue issubstituted in at least one position selected from the group consistingof position 2, 4, 36, 38, 40, 44, 46, 47, 48, 49, 64, 66, 68, 69, 71, 87and 98 of the light chain as numbered according to Kabat.
 16. The heavychain of claim 12, wherein the framework residue is substituted in atleast one position selected from the group consisting of position 2, 24,26, 27, 28, 29, 37, 39, 45, 47, 48, 67, 71, 73, 78, 91, 93, 94 and 103of the heavy chain as numbered according to Kabat.
 17. The heavy chainof claim 12, wherein the framework residue is substituted at a positionselected from the group consisting of position 24, 28, 29, 37, 48, 67,71, 73 and 78 of the heavy chain as numbered according to Kabat.
 18. Thelight chain of claim 11 or 13, wherein the framework residue isidentified by modeling the 12A11 light chain based on the solvedstructure of a murine immunoglobulin light chain that shares at least70% sequence identity with the 12A11 light chain.
 19. The light chain ofclaim 11 or 13, wherein the framework residue is identified by modelingthe 12A11 light chain based on the solved structure of a murineimmunoglobulin light chain that shares at least 80% sequence identitywith the 12A11 light chain.
 20. The light chain of claim 11 or 13,wherein the framework residue is identified by modeling the 12A11 lightchain based on the solved structure of a murine immunoglobulin lightchain that shares at least 90% sequence identity with the 12A11 lightchain.
 21. The heavy chain of claim 12 or 14, wherein the frameworkresidue is identified by modeling the 12A11 heavy chain based on thesolved structure of a murine immunoglobulin heavy chain that shares atleast 70% sequence identity with the 12A11 heavy chain.
 22. The heavychain of claim 12 or 14, wherein the framework residue is identified bymodeling the 12A11 heavy chain based on the solved structure of a murineimmunoglobulin heavy chain that shares at least 80% sequence identitywith the 12A11 heavy chain.
 23. The heavy chain of claim 12 or 14,wherein the framework residue is identified by modeling the 12A11 heavychain based on the solved structure of a murine immunoglobulin heavychain that shares at least 90% sequence identity with the 12A11 heavychain.
 24. A humanized immunoglobulin comprising the light chain of anyone of claims 1, 2, 5-7, 11, 13, 15 and 18-20, and the heavy chain ofany one of claims 3, 4, 8-10, 12, 14, 16-17 and 21-23, orantigen-binding fragment of said immunoglobulin.
 25. The immunoglobulinor antigen binding fragment of claim 24, which specifically binds tobeta amyloid peptide (Aβ) with a binding affinity of at least 10⁻⁷ M.26. The immunoglobulin or antigen binding fragment of claim 24, whichspecifically binds to beta amyloid peptide (Aβ) with a binding affinityof at least 10⁻⁸ M.
 27. The immunoglobulin or antigen binding fragmentof claim 24, which specifically binds to beta amyloid peptide (Aβ) witha binding affinity of at least 10⁻⁹ M.
 28. The immunoglobulin or antigenbinding fragment of claim 24, wherein the heavy chain isotype is γl. 29.The immunoglobulin or antigen binding fragment of claim 24, which bindsto soluble beta amyloid peptide (Aβ).
 30. The immunoglobulin or antigenbinding fragment of claim 24, which binds to aggregated beta amyloidpeptide (Aβ).
 31. The immunoglobulin or antigen binding fragment ofclaim 24, which mediates phagocytosis of beta amyloid peptide (Aβ). 32.The immunoglobulin or antigen binding fragment of claim 24, whichcrosses the blood-brain barrier in a subject.
 33. The immunoglobulin orantigen binding fragment of claim 24, which reduces beta amyloid peptide(Aβ) plaque burden in a subject.
 34. A humanized immunoglobulincomprising a humanized heavy chain and a humanized light chain, wherein(a) the humanized light chain comprises three complementaritydetermining regions (CDR1, CDR2 and CDR3) having amino acid sequencesfrom the corresponding complementarily determining regions of the mouse12A11 immunoglobulin light chain variable domain designated SEQ ID NO:2,and a variable region framework from a human light chain variable regionframework sequence provided that at least one framework residue selectedfrom the group consisting of a canonical residue, a vernier residue, apacking residue and a rare residue, is occupied by the same amino acidresidue present in the equivalent position of the mouse 12A11immunoglobulin light chain variable region framework; and (b) thehumanized heavy chain comprises three complementarity determiningregions (CDR1, CDR2 and CDR3) having amino acid sequences from thecorresponding complementarity determining regions of the mouse 12A11immunoglobulin heavy chain variable domain designated SEQ ID NO:4, and avariable region framework from a human heavy chain variable regionframework sequence provided that at least one framework residue selectedfrom a second group consisting of a canonical residue, a vernierresidue, a packing residue and a rare residue, is occupied by the sameamino acid residue present in the equivalent position of the mouse 12A11immunoglobulin heavy chain variable region framework; wherein thehumanized immunoglobulin specifically binds to beta amyloid peptide(“Aβ”) with a binding affinity of at least 10⁻⁷ M, wherein the 12A11immunoglobulin has the light chain with a variable domain designated SEQID NO:2 and the heavy chain with a variable domain designated SEQ IDNO:4.
 35. The humanized immunoglobulin of claim 34, wherein human lightchain variable region framework is from a kappa light chain variableregion.
 36. The humanized immunoglobulin of claim 34, wherein humanheavy chain variable region framework is from an IgG1 heavy chainvariable region.
 37. The humanized immunoglobulin of claim 34, whereinthe light chain variable region framework is from a human immunoglobulinlight chain having at least 70% sequence identity with light chainsequence of the 12A11 immunoglobulin.
 38. The humanized immunoglobulinof claim 34, wherein the heavy chain variable region framework is from ahuman immunoglobulin heavy chain having at least 70% sequence identitywith heavy chain sequence of the 12A11 immunoglobulin.
 39. The humanizedimmunoglobulin of claim 34, wherein the humanized light chain comprisescomplementarity determining regions that are identical to thecorresponding complementarity determining regions of the mouse 12A11heavy chain, and the humanized heavy chain comprises complementaritydetermining regions that are identical to the correspondingcomplementarity determining regions of the mouse 12A11 heavy chain. 40.A humanized antibody comprising the complementarity determining regions(CDR1, CDR2 and CDR3) of the 12A11 variable light chain sequence setforth as SEQ ID NO:2.
 41. A humanized antibody comprising thecomplementarity determining regions (CDR1, CDR2 and CDR3) of the 12A11variable heavy chain sequence set forth as SEQ ID NO:4.
 42. A humanizedantibody, or antigen-binding fragment thereof, which specifically bindsto beta amyloid peptide (Aβ), comprising a variable region comprisingcomplementarity determining regions (CDRs) corresponding to CDRs fromthe mouse 12A11 antibody.
 43. A humanized immunoglobulin having thevariable light chain amino acid sequence set forth as SEQ ID NO:6 andhaving a variable heavy chain amino acid sequence selected from thegroup consisting of SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10.
 44. Achimeric immunoglobulin comprising variable region sequencesubstantially as set forth in SEQ ID NO:2 or SEQ ID NO:4, and constantregion sequences from a human immunoglobulin.
 45. A method of preventingor treating an amyloidogenic disease in a patient, comprisingadministering to the patient an effective dosage of the humanizedimmunoglobulin of any one of claims 24-43.
 46. A method of preventing ortreating Alzheimer's disease in a patient, comprising administering tothe patient an effective dosage of the humanized immunoglobulin of anyone of claims 24-43.
 47. The method of claim 46, wherein the effectivedosage of humanized immunoglobulin is at least about 1 mg/kg bodyweight.
 48. The method of claim 46, wherein the effective dosage ofhumanized immunoglobulin is at least about 10 mg/kg body weight.
 49. Apharmaceutical composition comprising the immunoglobulin of any one ofclaims 24-43 and a pharmaceutical carrier.
 50. An isolated polypeptidecomprising a fragment of SEQ ID NO:2 selected from the group consistingof amino acids 43-58 of SEQ ID NO:2, amino acids 74-80 of SEQ ID NO:2and amino acids 113-121 of SEQ ID NO:2.
 51. An isolated polypeptidecomprising amino acids 43-48 of SEQ ID NO:2, amino acids 74-80 of SEQ IDNO:2 and amino acids 113-121 of SEQ ID NO:2.
 52. An isolated polypeptidecomprising a fragment of SEQ ID NO:4 selected from the group consistingof amino acids 50-56 of SEQ ID NO:4, amino acids 71-86 of SEQ ID NO:4and amino acids 119-128 of SEQ ID NO:4.
 53. An isolated polypeptidecomprising amino acids 50-56 of SEQ ID NO:4, amino acids 71-86 of SEQ IDNO:4 and amino acids 119-128 of SEQ ID NO:4.
 54. An isolated polypeptidecomprising amino acids 1-131 of SEQ ID NO:2.
 55. An isolated polypeptidecomprising amino acids 1-139 of SEQ ID NO:4.
 56. An isolated polypeptidehaving at least 85% identity to amino acids 1-131 of SEQ ID NO:
 2. 57.An isolated polypeptide having at least 85% identity to amino acids1-139 of SEQ ID NO:
 4. 58. The isolated polypeptide of claim 56 or 57,wherein the polypeptide has at least 86% identity.
 59. The isolatedpolypeptide of claim 56 or 57, wherein the polypeptide has at least 87%identity.
 60. The isolated polypeptide of claim 56 or 57, wherein thepolypeptide has at least 88% identity.
 61. The isolated polypeptide ofclaim 56 or 57, wherein the polypeptide has at least 89% identity. 62.The isolated polypeptide of claim 56 or 57, wherein the polypeptide hasat least 90% identity.
 63. The isolated polypeptide of claim 56 or 57,wherein the polypeptide has at least 90% or more identity.
 64. A variantof a polypeptide comprising the amino acid sequence of SEQ ID NO:2, saidvariant comprising at least one conservative amino acid substitution,wherein the variant retains the ability to specifically bind betaamyloid peptide (Aβ) with a binding affinity of at least 10⁷ M⁻¹.
 65. Avariant of a polypeptide comprising the amino acid sequence of SEQ IDNO:4, said variant comprising at least one conservative amino acidsubstitution, wherein the variant retains the ability to direct specificbinding to beta amyloid peptide (Aβ) with a binding affinity of at least10⁷ M⁻¹.
 66. An isolated nucleic acid molecule encoding the light chainof any one of claims 1, 2, 5-7, 11, 13, 15 and 18-20.
 67. An isolatednucleic acid molecule encoding the heavy chain of any one of claims 3,4, 8-10, 12, 14, 16-17 and 21-23.
 68. An isolated nucleic acid moleculeencoding the polypeptide of any one of claims 46-65.
 69. An isolatednucleic acid molecule encoding the immunoglobulin of any one of claims21-44.
 70. An isolated nucleic acid molecule comprising the nucleotidesequence of SEQ ID NO: 1 or
 3. 71. A vector comprising the nucleic acidmolecule of any of claims 66-70.
 72. A host cell comprising the nucleicacid molecule of any of claims 66-70.
 73. A transgenic animal expressinga polypeptide encoded by the nucleic acid molecule of any of thepreceding claims.
 74. The transgenic animal of claim 73, wherein thepolypeptide is expressed in the milk of said animal.
 75. A method ofproducing an antibody, or fragment thereof, comprising culturing thehost cell of claim 72 under conditions such that the antibody orfragment is produced and isolating said antibody from the host cell orculture.
 76. A method of producing an antibody or antibody fragment,comprising a fragment of SEQ ID NO:2 selected from the group consistingof amino acids 43-58 of SEQ ID NO:2, amino acids 74-80 of SEQ ID NO:2and amino acids 113-121 of SEQ ID NO:2, said method comprising culturinga host cell comprising a nucleic acid molecule that encodes saidantibody or antibody fragment under conditions such that the antibody orantibody fragment is produced, and isolating said antibody or antibodyfragment from the host cell or culture.
 77. A method of producing anantibody or antibody fragment, comprising a fragment of SEQ ID NO:4selected from the group consisting of amino acids 50-56 of SEQ ID NO:4,amino acids 71-86 of SEQ ID NO:4 and amino acids 119-128 of SEQ ID NO:4,said method comprising culturing a host cell comprising a nucleic acidmolecule that encodes said antibody or antibody fragment, underconditions such that the antibody or antibody fragment is produced, andisolating said antibody or antibody fragment from the host cell orculture.
 78. A method for identifying residues amenable to substitutionin a humanized 12A11 immunoglobulin variable framework region,comprising modeling the three-dimensional structure of the 12A11variable region based on a solved immunoglobulin structure and analyzingsaid model for residues capable of affecting 12A11 immunoglobulinvariable region conformation or function, such that residues amenable tosubstitution are identified.
 79. Use of the variable region sequence setforth as SEQ ID NO:2 or SEQ ID NO:4, or any portion thereof, inproducing a three-dimensional image of a 12A11 immunoglobulin, 12A11immunoglobulin chain, or domain thereof.
 80. A methods of imagingamyloid deposits in the brain of a patient comprising administering tothe patient an agent that specifically binds to Aβ, and detecting theantibody bound to Aβ.
 81. The method of claim 80, wherein the agent isan antibody comprising a light chain variable sequence as set forth inSEQ ID NO:2 and a heavy chain variable region sequence as set forth inSEQ ID NO:4, or an antigen-binding fragment of said antibody.
 82. Themethod of claim 80, wherein the antigen-binding fragment is a Fabfragment.
 83. A kit for imaging according to the method of any one ofclaims 80-82 including instructions for use.
 84. A method of treating anamyloidogenic disease comprising administering to a patient having saidamyloidogenic disease, a nucleic acid molecule that encodes animmunoglobulin light chain comprising the CDRs of the amino acidsequence of SEQ ID NO:2 and a nucleic acid molecule that encodes animmunoglobulin heavy chain that comprises the CDRs of the amino acidsequence of SEQ ID NO:4, under conditions such that said immunoglobulinchains are expressed, such that a beneficial therapeutic response insaid patient is generated.
 85. A method of treating an amyloidogenicdisease comprising administering to a patient having said amyloidogenicdisease, a nucleic acid molecule that encodes an immunoglobulin lightchain comprising the amino acid sequence of SEQ ID NO:7 and a nucleicacid molecule that encodes an immunoglobulin heavy chain that comprisesthe amino acid sequence of SEQ ID NO:10 or the amino acid sequence ofany one of SEQ ID NOs: 13-31, under conditions such that saidimmunoglobulin chains are expressed, such that a beneficial therapeuticresponse in said patient is generated.
 86. A method of preventing ortreating a disease associated with amyloid deposits of Aβ in the brainof a patient, comprising administering to the patient an effectivedosage of the humanized immunoglobulin of any one of the precedingclaims.
 87. The method of claim 86, wherein the disease is characterizedby cognitive impairment.
 88. The method of claim 86, wherein the diseaseis Alzheimer's disease.
 89. The method of claim 86, wherein the diseaseis Down's syndrome.
 90. The method of claim 86, wherein the disease ismild cognitive impairment.
 91. The method of claim 86, wherein theantibody is of human isotype IgG1.
 92. The method of any of thepreceding claims, wherein the patient is human.
 93. The method of anyone of claims 86-92, wherein after administration the antibody binds toan amyloid deposit in the patient and induces a clearing responseagainst the amyloid deposit.
 94. The method of claim 93, wherein theclearing response is an Fc receptor-mediated phagocytosis response. 95.The method of claim 93 or 94, further comprising monitoring the clearingresponse.
 96. The method of any one of claims 86-92, wherein afteradministration the antibody binds to soluble Aβ in the patient.
 97. Themethod of any one of claims 86-92, wherein after administration theantibody binds to soluble Aβ in the serum, blood or cerebrospinal fluidof the patient.
 98. The method of any of one of claims 86-97, whereinthe patient is asymptomatic.
 99. The method of any one of claims 86-98,wherein the patient is under 50 years of age.
 100. The method of any oneof claims 86-99, wherein the patient has an inherited risk factorindicating susceptibility to Alzheimer's disease.
 101. The method of anyone of claims 86-100, wherein the dosage of antibody is at least 1 mg/kgbody weight of the patient.
 102. The method of any one of claims 86-100,wherein the dosage of antibody is at least 10 mg/kg body weight of thepatient.
 103. The method of any one of claims 86-102, wherein theantibody is administered with a carrier as a pharmaceutical composition.104. The method of any one of claims 77-103, wherein the antibody isadministered intraperitoneally, orally, intranasally, subcutaneously,intramuscularly, topically or intravenously.
 105. A method for reducingplaque burden in a subject in need thereof comprising administering tothe patient an effective dosage of the humanized immunoglobulin of anyone of the preceding claims.
 106. The method of claim 105, wherein afteradministration the antibody binds to an amyloid deposit in the patientand induces a clearing response against the amyloid deposit.
 107. Themethod of claim 106, wherein the clearing response is an Fcreceptor-mediated phagocytosis response.
 108. The method of claim 106 or107, further comprising monitoring the clearing response.
 109. Themethod of claim 105, wherein after administration the antibody binds tosoluble Aβ in the patient.
 110. The method of claim 105, wherein afteradministration the antibody binds to soluble Aβ in the serum, blood orcerebrospinal fluid of the patient.
 111. A humanized immunoglobulin ofany one of the preceding claims, which comprises a Fc region having analtered effector function.
 112. A method for reducing neuritic burden ina subject in need thereof comprising administering to the subject aneffective dose of a humanized 12A11 antibody which binds to an epitopewithin amino acids 3-7 of beta amyloid peptide (Aβ), wherein theantibody is of the IgG1 isotype.
 113. The method of claim 112, whereinthe subject has an amyloidogenic disease.
 114. The method of claim 113,wherein the amyloidogenic disease is Alzheimer's disease.
 115. A methodfor treating an amyloidogenic disease in a patient comprisingadministering to the patient an effective dose of a humanized 12A11antibody capable of reducing beta amyloid peptide (Aβ) burden andneuritic dystrophy in the patient, wherein the antibody binds to anepitope within amino acids 3-7 of Aβ and is of the IgG1 isotype. 116.The method of claim 115, wherein the amyloidogenic disease isAlzheimer's disease.
 117. A method for reducing beta amyloid peptide(Aβ) burden and neuritic dystrophy in a mammal comprising administeringto the mammal an effective dose of a humanized 12A11 antibody capable ofreducing beta amyloid peptide (Aβ) burden and neuritic dystrophy,wherein the antibody binds to an epitope within amino acids 3-7 of Aβand is of the IgG1 isotype.
 118. The method of claim 117, wherein themammal is a human.
 119. The method of claim 117 or 118, wherein themammal has an amyloidogenic disease.
 120. The method of claim 119,wherein the amyloidogenic disease is Alzheimer's disease.
 121. Atherapeutic composition comprising a humanized 12A11 antibody of theIgG1 isotype which binds to an epitope within amino acids 3-7 of betaamyloid peptide (Aβ), wherein the antibody is capable of reducingneuritic burden in a subject.
 122. A method for treating anamyloidogenic disease in a patient comprising administering to thepatient an effective dose of a humanized 12A11 antibody which binds tosoluble beta amyloid peptide (Aβ) and reduces neuritic dystrophy in thepatient, wherein the antibody binds to an epitope within amino acids 3-7of Aβ and is of the IgG1 isotype.
 123. The method of claim 122, whereinthe amyloidogenic disease is Alzheimer's disease.
 124. A therapeuticcomposition comprising a humanized 12A11 antibody of the IgG1 isotypewhich binds to soluble beta amyloid peptide (Aβ), wherein the antibodybinds to an epitope within amino acids 3-7 of Aβ and is capable ofreducing neuritic burden in a subject.